Uses of Wnt polypeptides

ABSTRACT

Uses for Wnt polypeptides in hematopoiesis are disclosed. In particular, in vitro and in vivo methods for enhancing proliferation, differentiation or maintenance of a hematopoietic stem/progenitor cell using a Wnt polypeptide, and optionally another cytokine, are described.

CROSS REFERERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) of U.S.provisional application No. 60/024,068, having an effective filing dateof Aug. 16, 1996.

FIELD OF THE INVENTION

This application relates to uses for Wnt polypeptides ("Wnts"). Inparticular, the invention relates to uses for a Wnt polypeptide forenhancing the proliferation, differentiation and/or maintenance ofprimitive hematopoietic cells, e.g., hematopoietic stem/progenitorcells.

BACKGROUND OF THE INVENTION

A. HEMATOPOIESIS

The process of blood cell formation whereby red and white blood cellsare replaced through the division of cells located in the bone marrow iscalled hematopoiesis. For a review of hematopoiesis see Dexter andSpooncer (Ann. Rev. Cell Biol., 3:423-441 [1987]).

There are many different types of blood cells which belong to distinctcell lineages. Along each lineage, there are cells at different stagesof maturation. Mature blood cells are specialized for differentfunctions. For example, erythrocytes are involved in O₂ and CO₂transport; T and B lymphocytes are involved in cell and antibodymediated immune responses, respectively; platelets are required forblood clotting; and the granulocytes and macrophages act as generalscavengers and accessory cells. Granulocytes can be further divided intobasophils, eosinophils, neutrophils and mast cells.

Each of the various blood cell types arises from pluripotent ortotipotent stem cells which are able to undergo self-renewal or giverise to progenitor cells or Colony Forming Units (CFU) that yield a morelimited array of cell types. As stem cells progressively lose theirability to self-renew, they become increasingly lineage restricted. Ithas been shown that stem cells can develop into multipotent cells(called "CFC-Mix" by Dexter and Spooncer, supra). Some of the CFC-Mixcells can undergo renewal whereas others lead to lineage-restrictedprogenitors which eventually develop into mature myeloid cells (e.g.,neutrophils, megakaryocytes, macrophages, basophils and erythroidcells). Similarly, pluripotent stem cells are able to give rise to PreBand PreT lymphoid cell lineages which differentiate into mature B and Tlymphocytes, respectively. Progenitors are defined by their progeny,e.g., granulocytelmacrophagecolony-forming progenitor cells (GM-CFU)differentiate into neutrophils or macrophages; primitive erythroidburst-forming units (BFU-E) differentiate into erythroid colony-formingunits (CFU-E) which give rise to mature erythrocytes. Similarly, theMeg-CFU, Eos-CFU and Bas-CFU progenitors are able to differentiate intomegakaryocytes, eosinophils and basophils, respectively.

The number of pluripotent stem cells in the bone marrow is extremely lowand has been estimated to be in the order of about one per 10,000 to oneper 100,000 cells (Boggs et al., J. Clin. Inv., 70:242 [1982] andHarrison et al., PNAS, 85; 822 [1988]). Accordingly, characterization ofstem cells has been difficult. Therefore, various protocols forenriching pluripotent stem cells have been developed. See, for example,Matthews et al., Cell, 65:1143-1152 [1991]; WO 94/02157; Orlic et al.,Blood, 82(3 :762-770 [1993]; and Visseret al., Stem Cells, 11Suppl.(2):49-55 [July 1993].

Various lineage-specific factors have been demonstrated to control cellgrowth, differentiation and the functioning of hematopoietic cells.These factors or cytokines include the interleukins (e.g., IL-3),granulocyte-macrophagecolony-stimulatingfactor (GM-CSF), macrophagecolony-stimulating factor (M-CSF), granulocyte colony-stimulating factor(M-CSF), erythropoietin (Epo), Iymphotoxin, steel factor (SLF), tumornecrosis factor (TNF) and gamma-interferon. These growth factors have abroad spectrum of activity, from generalized to lineage-specific rolesin hematopoiesis, or a combination of both. For example, IL-3 appears toact on multipotent stem cells as well as progenitors restricted to thegranulocytelmacrcphage, eosinophil, megakaryocyte, erythroid or mastcell lineages. On the other hand, Epo generally acts on fairly matureerythroid progenitor cells.

B. THE HEMATOPOIETIC ENVIRONMENT AND EMBRYOGENESIS

The capacity of the hematopoietic stem cells to provide for the lifelongproduction of all blood lineages is accomplished by a balance betweenthe plasticity of the stem cell, that is the production of committedprogenitors cells which generate specific blood lineages, and thereplication of the stem cell in the undifferentiated state(self-renewal). The mechanisms regulating hematopoietic stem cells'plasticity and self-renewal in vivo have been difficult to define.However, the major contributory factors represent a combination of cellintrinsic and environmental influences (Morrison et al., Proc. Natl.Acad. Sci. USA, 92: 10302-10306 [1995]). The importance of thehematopoieticmicroenvironmenthas been established through the use oflong term bone marrow culture systems where hematopoieticcells culturedon stroma allow for the maintenance of HSCs, albeit at low frequencies(Fraser et al., Proc. Natl. Acad. Sci. USA, 89: 1968-1972, [1992];Wineman et al., Blood, 81: 365-372 [1993]).

The demonstration of hematopoietic cell maintenance in culture has ledto efforts to identify candidate `stem cell` factors. The role ofhematopoietic cytokines in stem cell maintenance has been studied bydirect addition of purified factors to in vitro cultures of stem cellpopulations followed by transplantation of the cultured cells (Muench etal., Blood, 81: 3463-3473 [1993]; Wineman et al., supra [1993]; Rebel etal., Blood, 83: 128-136 [1994]). Most of the known `early-acting`cytokines such as IL-3, IL-6, and KL have been shown to stimulateproliferation of more committed progenitor cells while concurrentlyallowing maintenance, but not expansion, of cells capable of long-termmultilineage repopulation (reviewed in Williams, Blood, 81(12):3169-3172[1993]; Muller-Sieburg and Deryugina, Stem Cells, 13: 477-486 [1995]).While these data indicate that the cells' plasticity and repopulatingfunction may be preserved by cytokine treatment, the molecules thatpromote self-renewal of these pluripotent cells remain unknown.

Transplantation studies have shown that the signals that regulate fatepluripotent stem cells may be similar in the embryo and adult bonemarrow. Cells from the day 11 fetal liver, yolk sac, oraorta/gonad/mesonephros (AGM) region can repopulate the adult marrow andappropriately respond to extrinsic cues to sustain long-termmultilineage hematopoiesis (Muller et al., Immunity, 1:291-301 [1994]).Although embryonic hematopoiesis is largely devoted to the erythroidlineage, the embryonic microenvironment clearly contributes to themaintenance of pluripotent stem cells in the undifferentiatedstate.These cell populations are cycling during embryogenesis (Zeigler et al.,supra [1994]; Morrison et al., supra [1995]; Rebel et al., Blood, 87:3500-3507 [1996]).

In mammals, hematopoietic precursors arise in the extraembryonic andventral mesoderm, yolk sac, or AGM region (Dzierzakand Medvinsky TrendsGenet., 11: 359-366 [1995]; Zon, Blood, 86: 2876-2891 [1995]). Inamphibian embryos, the equivalent regions are the ventral blood islandmesoderm and the dorsal lateral plate mesoderm (reviewed in Kessler andMelton, Science, 266: 596-604 [1994]; Zon, supra 1995; Tam and Quinlan,Curr. Biol., 6: 104-106 [1996]). Secreted factors that potentiallyregulate cell fate determination of ventral mesoderm in Xenopus includeWnts, FGFs, and BMP-4 (reviewed in Christian and Moon, Bio Essays, 15:135-140 [1993a]; Zon, supra [1995]). Embryonic expression of XWnt-8(Christian and Moon, Genes Dev., 7: 13-28 [1993b]) and XWnt-11(Ku andMelton, Development, 119: 1161-1173 1993]) is localized to the area ofprospective ventral and lateral mesoderm and XWnt-8 expression can beinduced by ventralizing factors such as FGFs and BMP-4.

C. THE WNTS GENE FAMILY

Wnts are encoded by a large gene family whose members have been found inround worms, insects, cartilaginous fish and vertebrates (Sidow, 1994).Wnts are thought to function in a variety of developmental andphysiological processes since many diverse species have multipleconserved Wnt genes (McMahon, Trends Genet., 8: 236-242 [1992]; Nusseand Varmus, Cell, 69: 1073-1087 [1992]). Wnt genes encode secretedglycoproteins that are thought to function as paracrine or autocrinesignals active in several primitive cell types (McMahon, supra [1 992];Nusse and Varmus, supra [1992]). The Wnt growth factor family includesmore than 10 genes identified in the mouse (Wnt-1, 2, 3a, 3b, 4, 5a, 5b,6, 7a 7b, 8a, 8b, 10b, 11, 12) (see, e.g., Gavin et al., Genes Dev., 4:2319-2332 [1990]; Lee et al., Proc. Natl. Acad. Sci. USA, 92: 2268-2272;Christiansen et al., Mech. Dev. 51: 341-350 [1995]) and at least 7 genesidentified in the human (Wnt-1, 2, 3,4, 5a, 7a and 7b) by cDNA cloning(see, e.g., Vant Veer et al., Mol. Cell. Biol., 4: 2532-2534 [1984]).The Wnt-1 proto-oncogene (int-1) was originally identified from mammarytumors induced by mouse mammary tumor virus (MMTV) due to an insertionof viral DNA sequence (Nusse and Varmus, Cell, 31: 99-109 [1982]). Inadult mice, the expression level of Wnt-1 mRNA is detected only in thetestis during later stages of sperm development. Wnt-1 protein is about42 KDa and contains an amino terminal hydrophobic region, which mayfunction as a signal sequence for secretion (Nusse and Varmus, supra).The expression of Wnt-2/irp is detected in mouse fetal and adult tissuesand its distribution does not overlap with the expression pattern forWnt-1. Wnt-3 is associated with mouse mammary tumorigenesis. Theexpression of Wnt-3 in mouse embryos detected in the neural tubes and inthe limb buds. Wnt-5a transcripts are detected in the developing fore-and hind limbs at 9.5 through 14.5 days and highest levels areconcentrated in apical ectoderm at the distal tip of limbs (Nusse andVarmus, supra [1992]. Recently, a Wnt growth factor, termed Wnt-x, wasdescribed (PCT/US94/14708; W095/17416) along with the detection of Wnt-xexpression in bone tissues and in bone-derived cells. Also described wasthe role of Wnt-x in the maintenance of mature osteoblasts and the useof the Wnt-x growth factor as a therapeutic agent or in the developmentof other therapeutic agents to treat bone-related diseases.

Wnts may play a role in local cell signaling. Biochemical studies haveshown that much of the secreted Wnt protein can be found associated withthe cell surface or extracellular matrix rather than freely diffusiblein the medium (Papkoff and Schryver, Mol. Cell. Biol., 10: 2723-2730[1990]; Bradley and Brown, EMBO J., 9: 1569-1575 [1990]).

Studies of mutations in Wnt genes have indicated a role for Wnts ingrowth control and tissue patterning. In Drosophila, wingless (wg)encodes a Wnt gene (Rijsenijk et al., Cell. 50: 649-657 [1987]) and wgmutations alter the pattern of embryonic ectoderm, neurogenesis, andimaginal disc outgrowth (Morata and Lawrence, Dev. Biol., 56: 227-240[1977]; Baker, Dev. Biol., 125: 96-108 [1988]; Klingensmith and Nusse,Dev. Biol., 166: 396-414[1994]). In Caenorhabditis elegans, lin-44encodes a Wnt which is required for asymmetric cell divisions (Hermanand Horvitz, Development, 120: 1035-1047 [1994]). Knock-out mutations inmice have shown Wnts to be essential for brain development (McMahon andBradley, Cell, 62: 1073-1085 [1990]; Thomas and Cappechi, Nature, 346:847-850 [1990]), and the outgrowth of embryonic primordia for kidney(Stark et al., Nature, 372: 679-683 [1994]), tail bud (Takada et al.,Genes Dev., 8: 174-189 [1994]), and limb bud (Parr and McMahon, Nature,374: 350-353 [1995]). Overexpression of Wnts in the mammary gland canresult in mammary hyperplasia (McMahon, supra (1992]; Nusse and Varmus,supra [1992]), and precocious alveolar development (Bradbury et al.,Dev. Biol., 170: 553-563 [1995]). A role for Wnts in mammalianhematopoiesis has not previously been suggested or considered.

Wnt-5a and Wnt-5b are expressed in the posterior and lateral mesodermand the extraembryonic mesoderm of the day 7-8 murine embryo (Gavin etal., supra [1990]). These embryonic domains contribute to the AGM regionand yolk sac tissues from which multipotent hematopoietic precursors andHSCs are derived (Dzierzak and Medvinsky, supra [1995]; Zon, supra[1995], Kanatsu and Nishikawa, Development, 122: 823-830 [1996]).Wnt-5a, Wnt-10b, and other Wnts have been detected in limb buds,indicating possible roles in the development and patterning of the earlybone microenvironment as shown for Wnt-7b (Gavin et al., supra [1990];Christiansen et al., Mech. Devel., 51: 341-350 [1995]; Parr and McMahon,supra [1995]).

D. HEMATOPOIETIC DISEASES AND DISORDERS

Chemo- and radiation therapies cause dramatic reductions in blood cellpopulations in cancer patients. At least 500,000 cancer patients undergochemotherapy and radiation therapy in the US and Europe each year andanother 200,000 in Japan. Bone marrow transplantation therapy of valuein aplastic anemia, primary immunodeficiency and acute leukemia(following total body irradiation) is becoming more widely practiced bythe medical community. At least 15,000 Americans have bone marrowtransplants each year. Other diseases can cause a reduction in entire orselected blood cell lineages. Examples of these conditions includeanemia (including macrocytic and aplastic anemia); thrombocytopenia;hypoplasia; immune (autoimmune) thrombocytopenic purpura (ITP); and HIVinduced ITP.

Pharmaceutical products are needed which are able to enhancereconstitution of blood cell populations of these patients.

Accordingly, it is an object of the present invention to provide amethod for enhancing the proliferation and/or differentiation and/ormaintenance of primitive hematopoietic cells. Such a method may beuseful for enhancing repopulation of hematopoietic stem cells and thusmature blood cell lineages. This is desirable where a mammal hassuffered a decrease in hematopoietic or mature blood cells as aconsequence of disease, radiation or chemotherapy. This method is alsouseful for generating expanded populations of such stem cells and matureblood cell lineages from such hematopoietic cells ex vivo.

These and other objects will be apparent to the ordinary artisan uponconsideration of the specification as a whole.

SUMMARY OF THE INVENTION

In one aspect, the present invention pertains to the discovery hereinthat Wnt polypeptides ("Wnts"), such as Wnt-5a, play a role inhematopoiesis. In another aspect, the present invention is based on theobservation that such Wnts function as hematopoietic regulatory factorsand are able to directly stimulate the proliferation of hematopoieticstem cells, trigger the formation of multicellular aggregates or `foci`of primitive blast cells, and expand the total number of multipotentialcolony forming cells via receptor signalling. Wnts appeared to directlyact at the level of the early hematopoietic precursor (i.e.,hematopoieticstem/progenitorcells). Such an expanded stem cellpopulation can serve as the source of cells for myelopoiesis,erythropoiesis (e.g., splenic erythropoiesis) and lymphopoiesis.Accordingly, Wnts can be used to stimulate proliferation and/ordifferentiation and/or maintenance of hematopoietic stem/progenitorcells either in vitro or in vivo (e.g., for treating hematopoieticdiseases or disorders).

Thus, the invention provides a method for enhancing proliferation,differentiation and/or maintenance of a cell with a Wnt polypeptidecomprising the step of contacting the cell with an amount of Wntpolypeptide which is effective for stimulating proliferation and/ordifferentiation and/or maintenance (e.g., survival) of the cell. Inpreferred embodiments, the cell which is exposed to the Wnt polypeptideis a hematopoietic precursor, e.g., a hematopoietic stem/progenitorcell. For example, the Wnt polypeptide may be Wnt-1, Wnt-5a or Wnt-10b.For in vivo use, the Wnt polypeptide of choice may be a long half-lifederivative of, for example, a Wnt-5a polypeptide such as Wnt-5aimmunoglobulin chimera and/or Wnt-5a polypeptide modified with anonproteinaceous polymer, such as polyethylene glycol (PEG). The methodcontemplated herein may lead to an increase in the proliferation and/ordifferentiation of lymphoid, myeloid and/or erythroid blood celllineages from the maintained or expanded hematopoietic stem/progenitorcell population and encompasses both in vitro and in vivo methods. Forin vitro uses, the cell stimulated by Wnts may be present in cellculture. As to in vivo methods, the cell may be present in a mammal,especially a human (e.g., one who is suffering from decreased bloodlevels and who could benefit from an increase in various blood cells).Potential patients include those who have undergone chemo- or radiationtherapy, or bone marrow transplantation therapy. Thus, the inventionprovides a method for repopulating blood cells (e.g., erythroid, myeloidand/or lymphoid blood cells) in a mammal comprising administering to themammal a therapeutically effective amount of Wnt polypeptide.

Mammals which may benefit from an enhancement of lymphopoiesis includethose predisposed to, or suffering from, any one or more of thefollowing exemplary conditions: lymphocytopenia; lymphorrhea;lymphostasis; immunodeficiency (e.g., HIV and AIDS); infections(including, for example, opportunistic infections and tuberculosis(TB)); lupus; and other disorders characterized by lymphocytedeficiency. An effective amount of the Wnt polypeptide can be used in amethod of immunopotentiation or to improve immune function in a mammal.

Diseases or disorders in which an increase in erythropoiesis may bebeneficial include, but are not limited to: erythrocytopenia;erthrodegenerative disorders; erythroblastopenia; leukoerythroblastosis;erythroclasis; thalassemia; and anemia (e.g., hemolytic anemia, such asacquired, autoimmune, or microangiopathichemolytic anemia; aplasticanemia; congenital anemia, e.g., congenital dyserythropoietic anemia,congenital hemolytic anemia or congenital hypoplastic anemia;dyshemopoietic anemia; Faconi's anemia; genetic anemia; hemorrhagicanemia; hyperchromic or hypochromic anemia; nutritional, hypoferric, oriron deficiency anemia; hypoplastic anemia; infectious anemia; leadanemia; local anemia; macrocytic or microcytic anemia; malignant orpernicious anemia; megaloblastic anemia; molecular anemia; normocyticanemia; physiologic anemia; traumatic or posthemorrhagic anemia;refractory anemia; radiation anemia; sickle cell anemia; splenic anemia;and toxic anemia).

An increase in myelopoiesis may be beneficial in any of theabove-mentioned diseases or disorders as well as the following exemplaryconditions; myelofibrosis; thrombocytopenia; hypoplasia; disseminatedintravascularcoagulation (DIC); immune (autoimmune)thrombocytopeniopurpura (ITP); HIV inducted ITP; myelodysplasia; thrombocytotic diseasesand thrombocytosis.

The method may further involve the step of exposing hematopoieti cells(whether they be in cell culture or in a mammal) to one or more othercytokines (e.g., lineage-specificcytokines) and this may lead to asynergistic enhancement of the proliferation and/or differentiation ofthe cells. Exemplary cytokines include thrombopoietin (TPO);erythropoietin (EPO); macrophage-colony stimulating factor (M-CSF);granulocyte-macrophage CSF (GM-CSF); granulocyte-CSF (G-CSF);interleukin-1 (IL-1); IL-1a; IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9; IL-11; IL10; IL-12; leukemia inhibitoryfactor (LIF) or kit ligand(KL). In this embodiment, exposure to the cytokine may proceed, occursimultaneously with, or follow, exposure to the Wnt polypeptide.Preferably, the Wnt polypeptide and one or more further cytokines areadministered simultaneously to the patient (where the method is an invivo one) and, optionally, are combined to form a pharmaceuticalcomposition.

For use in the above methods, the invention also provides an article ofmanufacture, comprising: a container, a label on the container, and acomposition comprising an active agent within the container, wherein thecomposition is effective for enhancing proliferation and/ordifferentiation and/or maintenance of hematopoietic stem/progenitorcells in a mammal, the label on the container indicates that thecomposition can be used for enhancing proliferation and/ordifferentiation and/or maintenance of those cells and the active agentin the composition is a Wnt polypeptide. Optionally, the article ofmanufacture includes one or more further containers which hold furthercytokine(s) in a packaged combination with the container holding the Wntpolypeptide.

In another embodiment, an effective amount of the Wnt polypeptide may beused to improve engraftment in bone marrow transplantation or tostimulate mobilization and/or expansion of hematopoietic stem cells in amammal prior to harvesting hematopoietic progenitors from the peripheralblood thereof.

In addition to the above, the invention provides isolated nucleic acidmolecules, expression vectors and host cells encoding a Wnt polypeptidewhich can be used in the recombinant production of Wnts as describedherein. The isolated nucleic acid molecules and vectors are also usefulfor gene therapy applications to treat patients, for example, toincrease the number of cells expressing a Wnt polypeptide and increaseWnt responsiveness. In addition, anti-Wnt antibodies, in particular,neutralizing antibodies to Wnts, are useful for the treatment ofdisorders, stem cell tumors and other tumors at sites of Wnt expression,including those tumors characterized by overexpression of Wnts.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing Wnts promotion of cell proliferation insuspension cultures of flASK cells. The fold expansions in cell numberfollowing culture for 7 days are shown. Cultures were initiated withflASK cells (5000/well), 25 ng/ml KL, and conditioned media (CM) from293 cells transfected with control plasmid, Wnt-1, Wnt-5a (gDWnt5aHis₆),or Wnt-10b. Assays were performed in duplicate and repeated in twoindependent experiments.

FIG. 2A, B and C are graphs showing Wnts promotion of enhanced foldexpansion and colony formation from flASK cells. FIG. 2A shows enhancedsurvival/proliferation of flASK cells following transduction with theWnt5a/LNL6 retrovirus. Transductions were initiated with 100,000cells/ml in IL-3, IL-6, and KL. The fold expansion for LNL6 orWnt5a/LNL6-treated cells was determined from cell counts at the end ofthe transduction period (48 hours) and repeated four times. FIG. 2Bshows that suspension culture of Wnt5a/LNL6 transduced cells for 7 daysresults in extensive expansion compared to LNL6-treated cultures. FIG.2C shows the colony formation from flASK cells following a 48 hourtransduction with LNL6 or Wnt5a/LNL6. Cells were plated in quadruplicatein myeloid methylcellulose, colony growth was evaluated after day 12 ofculture, and repeated in four independent experiments.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A search to uncover novel self-renewal factors from investigations onthe embryonic and fetal hematopoietic microenvironment has led to thediscovery that Wnt polypeptides comprise a novel class of stem cellregulators and directly stimulate the extensive proliferation and/ordifferentiation and/or maintenance of cultured hematopoieticstem/progenitor cells. Wnt polypeptides are thus useful in vivo or exvivo to enhance proliferation and/or differentiation and/or maintenanceof hematopoietic stem/progenitor cells, expand the population of thesecells and enhance repopulation of such cells and blood cells of multiplelineages in a mammal.

I. Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms "Wnts" or "Wnt gene product" or "Wnt polypeptide" when usedherein encompass native sequence Wnt polypeptides, Wnt polypeptidevariants, Wnt polypeptide fragments and chimeric Wnt polypeptides.Optionally, the Wnt polypeptide is not associated with nativeglycosylation. "Native glycosylation" refers to the carbohydratemoieties which are covalently attached to Wnt polypeptidewhen it isproduced in the mammalian cell from which it is derived in nature.Accordindy, a human Wnt polypeptide produced in a non-human cell is anexample of a Wnts which is "not associated with native glycosylation".Sometimes, the Wnt polypeptide is unglycosylated (e.g., as a result ofbeing produced recombinantly in a prokaryote).

A "native sequence" polypeptide is one which has the same amino acidsequence as a polypeptide (e.g., Wnt polypeptide) derived from nature.Such native sequence polypeptides can be isolated from nature or can beproduced by recombinant or synthetic means. Thus, a native sequencepolypeptidecan have the amino acid sequence of naturallyoccurring humanpolypeptide, murine polypeptide, or polypeptide from any other mammalianspecies.

The term "native sequence Wnt polypeptide" includes those Wntpolypeptides from any animal species (e.g., human, murine, rabbit, cat,cow, sheep, chicken, procine, equine, etc.) as occurring in nature. Thedefinition specifically includes human Wnt polypeptides, Wnt-1, 2, 3, 4,5a, 7a and 7b and murine Wnt polypeptides, Wnt-1, 2, 3a, 3b, 4, 5a, 5b,6, 7a, 7b, 8a, 8b, 10b, 11 and 12. The term "native sequence Wntprotein" includes the native proteins with or without the initiatingN-terminal methionine (Met), and with or without the native signalsequence. The native sequence human and murine Wnt polypeptides known inthe art are from about 348 to about 389 amino acids long in theirunprocessed form reflecting variability (particularly at the poorlyconserved amino-terminus and several internal sites), contain 21conserved cysteines, and have the features of a secreted protein (see,e.g., Wnt polypeptides as in Gavin et al., supra; Lee et al., supra;Christiansen et al., supra; PCT/US94/14708 [WO 95/17416]). The molecularweight of a Wnt polypeptide is about 38-42 kD in a monomeric form.

A "variant" polypeptide means a biologicallyactive polypeptideas definedbelow having less than 100% sequence identity with a native sequencepolypeptide. Such variants include polypeptides wherein one or moreamino acid residues are added at the N- or C-terminus of, or within, thenative sequence; from about one to forty amino acid residues aredeleted, and optionally substituted by one or more amino acid residues;and derivatives of the above polypeptides, wherein an amino acid residuehas been covalently modified so that the resulting product has anon-naturally occurring amino acid. Ordinarily, a biologically activeWnt variant will have an amino acid sequence having at least about 90%amino acid sequence identity with a native sequence Wnt polypeptide,preferably at least about 95%, more preferably at least about 99%.

A "chimeric"Wnt polypeptide is a polypeptide comprising a Wntpolypeptide or portion (e.g., one or more domains) thereof fused orbonded to heterologous polypeptide. The chimeric Wnt polypeptide willgenerally share at least one biological property in common with a nativesequence Wnt polypeptide, such as Wnt-5a. Examples of chimericpolypeptides include immunoadhesins and epitope tagged polypeptides.

The term "Wnt immunoadhesin" is used interchangeably with the expression"Wnt polypeptide-immunoglobulin chimera" and refers to a chimericmolecule that combines a portion of the Wnt polypeptide with animmunoglobulin sequence. The immunoglobulin sequence preferably, but notnecessarily, is an immunoglobulin constant domain. The immunoglobulinmoiety in the chimeras of the present invention may be obtained fromIgG1, IgG2, IgG3 or IgG4 subtypes, IgA, IgE, IgD or IgM, but preferablyIgG1 or IgG3.

The term "epitope tagged" when used herein refers to a chimericpolypeptide comprising a Wnt polypeptide or portion thereof fused to a"tag polypeptide". The tag polypeptide has enough residues to provide anepitope against which an antibody thereagainst can be made, yet is shortenough such that it does not interfere with biological activity of theWnt polypeptide. The tag polypeptide preferably also is fairly unique sothat the antibody thereagainst does not substantially cross-react withother epitopes. Suitable tag polypeptides generally have at least sixamino acid residues and usually between about 6-60 amino acid residues.

"Isolated" Wnt polypeptide means has been purified from a Wnts source orhas been prepared by recombinant or synthetic methods and issufficiently free of other peptides or proteins (1) to obtain at least15 and preferably 20 amino acid residues of the N-terminal or of aninternal amino acid sequence by using a spinning cup sequenatoror thebest commercially available amino acid sequenator marketed or asmodified by published methods as of the filing date of this application,or (2) to homogeneity by SDS-PAGE under non-reducing or reducingconditions using Coomassie blue or, preferably, silver stain.Homogeneity here means less than about 5% contamination with othersource proteins.

"Essentially pure" protein means a composition comprising at least about90% by weight of the protein, based on total weight of the composition,preferably at least about 95% by weight. "Essentially homogeneous"protein means a composition comprising at least about 99% by weight ofprotein, based on total weight of the composition.

"Biological property" when used in conjunction with either "Wntpolypeptide" or "isolated Wnt polypeptide" means having aneffectorfunction that is directly or indirectly caused or performed bynative sequence Wnt polypeptide, such as Wnt-5a. Effector functions ofnative sequence Wnt polypeptides include enhancement of differentiationand/or proliferation and/or maintenance of hematopoietic/progenitorcells(e.g., as determined in assays described in Examples 1 and 2). A"biologically active Wnt polypeptide" is one which possesses abiological property of native sequence Wnt polypeptide.

A "functional derivative" of a native sequence Wnt polypeptide is acompound having a qualitative biological property in common with anative sequence Wnt polypeptide. "Functional derivatives" include, butare not limited to, fragments of a native sequence and derivatives of anative sequence Wnt polypeptide and its fragments, provided that theyhave a biological activity in common with a corresponding nativesequence Wnt polypeptide. The term "derivative" encompasses both aminoacid sequence variants of Wnt polypeptide and covalent modificationsthereof.

The phrase "long half-life" as used in connection with Wnt polypeptides,concerns Wnt derivatives having a longer plasma half-life and/or slowerclearance than a corresponding native sequence Wnt polypeptide. The longhalf-life derivatives preferably will have a half-life at least about1.5-times longer than a native Wnt polypeptide; more preferably at leastabout 2-times longer than a native Wnt polypeptide, more preferably atleast about 3-times longer than a native Wnt polypeptide.

"Percent amino acid sequence identity" is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the residues in the native sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. None of N-terminal,C-terminal, or internal extensions, deletions, or insertions into thecandidate sequence shall be construed as affecting sequence identity orhomology.

The expression "control sequences" refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, aribosome binding site, and possibly, other as yet poorly understoodsequences. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, "operably linked"means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adapters or linkers are used in accordancewith conventional practice.

As used herein, the expressions "cell," "cell line," and "cell culture"are used interchangeablyand all such designationsinclude progeny. Thus,the words "transformants" and "transformed cells" or "transfectants" and"transfected cells" include the primary subject cell and culturesderived therefrom without regard for the number of transfers. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Mutant progeny thathave the same function or biological activity as screened for in theoriginally transformed cell are included. Where distinct designationsare intended, it will be clear from the context.

The term "antibody" is used in the broadest sense and specificallycovers monoclonal antibodies, antibody compositionswith polyepitopicspecificity, bispecific antibodies, diabodies, and single-chainmolecules, as well as antibody fragments (e.g., Fab, F(ab')₂, and Fv),so long as they exhibit the desired biological activity.

The term "monoclonal antibody" as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier "monoclonal"indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohleret al., Nature 256:495 [1975], or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567 (Cabilly etal.)). The "monoclonal antibodies" may also be isolated from phageantibody libraries using the techniques in Clackson et al., Nature352:624-628 [1991] and Marks et al., J. Mol. Biol. 222:581-597 [1991],for example.

The monoclonal antibodies herein specifically include "chimeric"antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibitthe desiredbiological activity (Cabilly et al., supra; Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855 [1984]).

"Humanized" forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab, F(ab)₂ or otherantigen-bindirg subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiesmay comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance. In general,the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulinsequence. The humanized antibody optimally also will comprise at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. For further details, see Jones et al., Nature321:522-525[1986]; Riechmann et al., Nature 332:323-327[1988]; andPresta, Curr. Op. Struct. Biol 2:593-596 [1992]. The humanized antibodyincludes a Primatized® antibody wherein the antigen-binding region ofthe antibody is derived from an antibody produced by immunizing macaquemonkeys with the antigen of interest.

"Non-immunogenicin a human" means that upon contacting the polypeptideof interest in a physiologically acceptable carrier and in atherapeutically effective amount with the appropriate tissue of a human,no state of sensitivityor resistanceto the polypeptide of interest isdemonstrated upon the second administration of the polypeptide ofinterest after an appropriate latent period (e.g., 8 to 14 days).

The phrase "enhancing proliferation of a cell" encompasses the step ofincreasing the extent of growth and/or reproduction of the cell relativeto an untreated cell either in vitro or in vivo. An increase in cellproliferation in cell culture can be detected by counting the number ofcells before and after exposure to a molecule of interest. The extent ofproliferation can be quantified via microscopicexamination of the degreeof confluency. Cell proliferation can also be quantified using athymidine incorporation assay.

By "enhancing differentiation of a cell" is meant the act of increasingthe extent of the acquisition or possession of one or morecharacteristics or functions which differ from that of the original cell(i.e., cell specialization). This can be detected by screening for achange in the phenotype of the cell (e.g., identifying morphologicalchanges in the cell and/or surface markers on the cell).

By "enhancing survival or maintenance of a cell" encompasses the step ofincreasing the extent of the possession of one or more characteristicsor functions which are the same as that of the original cell (i.e., cellphenotype maintenance). This can be detected by screening for themaintenance of the cell's phenotype (e.g., blast cell phenotype as inExample 2).

A "hematopoieticstem/progenitorcell" or "primitive hematopoieticcell" isone which is able to differentiate to form a more committed or matureblood cell type.

A "hematopoieticstem cell" or "stem cell" is one which is specificallycapable of long-term engraftment of a lethally irradiated host.

"Lymphoid blood cell lineages" are those hematopoietic precursor cellswhich are able to differentiate to form lymphocytes (B-cells orT-cells). Likewise, "lymphopoeisis" is the formation of lymphocytes.

"Erythroid blood cell lineages" are those hematopoietic precursor cellswhich are able to differentiate to form erythrocytes (red blood cells)and "erythropoeisis" is the formation of erythrocytes.

The phrase "myeloid blood cell lineages", for the purposes herein,encompasses all hematopoietic precursor cells, other than lymphoid anderythroid blood cell lineages as defined above, and "myelopoiesis"involves the formation of blood cells (other than lymphocytes anderythrocytes).

A "CD34⁺ cell population" is enriched for hematopoietic stem cells. ACD34⁺ cell population can be obtained from umbilical cord blood or bonemarrow, for example. Human umbilical cord blood CD34⁺ cells can beselected for using immunomagnetic beads sold by Miltenyi (California),following the manufacturer's directions.

An "AA4⁺ cell population" is enriched for hematopoietic stem cells. AnAA4⁺ cell population can be obtained from fetal liver for example. AA4⁺cells can be selected by immunoadherent panning, for example, with anantibody such as AA4.1.

A "Lin^(l0) Sca⁺ ", "cell population" or "AA4⁺ Sca⁺ cell population" isenriched for hematopoietic stem cells. Such populations can be obtainedfrom bone marrow or fetal liver, respectfully, for example. Lin^(lo)Sca⁺ cells or AA4⁺ Sca⁺ cells can be selected by cell sorting afterstaining with an antibody to the Sca-1 antigen (Ly6A/E), for example,the Ly6A/E phycoerythrin conjugate from Pharmingen (San Diego, Calif.).

A "flASK cell population" is highly enriched for hematopoietic stemcells from fetal liver. A flASK cell is a fetal liver, AA4⁺, Sca⁺, kit⁺cell. flASK cells can be selected by cell sorting after staining, forexample, with antibodies.

"Physiologically acceptable" carriers, excipients, or stabilizers areones which are nontoxic to the cell or mammal being exposed thereto atthe dosages and concentrations employed. Often the physiologicallyacceptable carrier is an aqueous pH buffered solution. Examples ofphysiologically acceptable carriers include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween®, Pluronic® or polyethylene glycol (PEG).

The term "cytokine" is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, growth factorsand traditional polypeptide hormones. Included among the cytokines aregrowth hormones such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen, OB protein; tumor necrosisfactor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -Il;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β and -γ; colony stimulatingfactors (CSFs) such asmacrophage-CSF(M-CS F); granulocyte-macrophage-CSF(GM-CSF); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; and otherpolypeptide factors including leukemia inhibitory factor (LIF) and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

A "lineage-specific cytokine" is one which acts on relatively committedcells in the hematopoietic cascadeand gives rise to an expansion inblood cells of a single lineage. Examples of such cytokines include EPO,TPO, and G-CSF.

"Treatment" refers to both therapeutictreatment and prophylacticorpreventative measures. Those in need of treatment include those alreadywith the disease or disorder as well as those in which the disease ordisorder is to be prevented.

"Mammal" for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

By "solid phase" is meant a non-aqueous matrix to which a reagent ofinterest (e.g., the Wnt polypeptide or an antibody thereto) can adhere.Examples of solid phases encompassed herein include those formedpartially or entirely of glass (e.g., controlled pore glass),polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinylalcohol and silicones. In certain embodiments, depending on the context,the solid phase can comprise the well of an assay plate; in others it isa purification column (e.g., an affinity chromatography column). Thisterm also includes a discontinuous solid phase of discrete particles,such as those described in U.S. Pat. No. 4,275,149.

II. Modes for Carrying Out the Invention

The present invention is based on the discovery of the uses of Wntpolypeptidesto enhance hematopoiesis. The experiments described hereindemonstrate that Wnts function as hematopoietic regulatory factors whichappear to play a role in enhancing proliferation, differentiation and/ormaintenance of hematopoietic cells. In particular, Wnts have been foundto be present in enriched human stem cell populations, and Wnts may beused to stimulate proliferation of hematopoietic stem cells/progenitorcells. Other uses for these polypeptides will be apparent from thefollowing discussion. A description follows as to how Wnt genes andpolypeptides may be prepared.

A. Preparation of Wnt Genes and Gene Products

Most of the discussion below pertains to recombinant production of Wntgenes and gene products by culturing cells transformed with a vectorcontaining Wnt polypeptide-encoding nucleic acid and recovering thepolypeptide from the cell culture.

1. Isolation of DNA Encoding Wnt Polypeptide

The DNA encoding Wnt polypeptide may be obtained from any cDNA libraryprepared from tissue believed to possess the Wnt polypeptide mRNA and toexpress it at a detectable level. Accordingly, Wnt polypeptide DNA canbe conveniently obtained from a cDNA library prepared from mammalianfetal liver or fetal brain. The Wnt polypeptide-encoding gene may alsobe obtained from a genomic library or by oligonucleotide synthesis.

Libraries are screened with probes (such as antibodies to the Wntpolypeptide, or oligonucleotides of about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures as described in chapters 10-12 of Sambrook etat, Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding Wnt polypeptide is to use PCR methodology as described insection 14 of Sambrook et at, supra.

A preferred method of practicing this invention is to use carefullyselected oligonucleotide sequences to screen cDNA librariesfrom varioushuman tissues, preferably human fetal liver. The oligonucleotidesequences selected as probes should be of sufficient length andsufficiently unambiguous that false positives are minimized.

The oligonucleotidemust be labeled such that it can be detected uponhybridizationto DNA in the library being screened. The preferred methodof labeling is to use ³² P-labeled ATP with polynucleotide kinase, as iswell known in the art, to radiolabel the oligonucleotide. However, othermethods may be used to label the oligonucleotide, including, but notlimited to, biotinylation or enzyme labeling.

Amino acid sequence variants of Wnt polypeptide are prepared byintroducing appropriate nucleotide changes into the Wnt polypeptide DNA,or by synthesis of the desired Wnt polypeptide. Such variants representinsertions, substitutions, and/or specified deletions of, residueswithin or at one or both of the ends of the amino acid sequence of anaturally occurring human Wnt polypeptide. Preferably, these variantsrepresent insertions and/or substitutions within or at one or both endsof the mature sequence, and/or insertions, substitutions and/orspecified deletions within or at one or both of the ends of the signalsequence of the Wnt polypeptide. Any combination of insertion,substitution, and/or specified deletion is made to arrive at the finalconstruct, provided that the final construct possesses the desiredbiological activity as defined herein. The amino acid changes also mayalter post-translational processes of the Wnt polypeptide, such aschanging the number or position of glycosylation sites, altering themembrane anchoring characteristics, and/or altering the intracellularlocation of the Wnt polypeptide by inserting, deleting, or otherwiseaffecting the leader sequence of the Wnt polypeptide.

Variations in the native sequence as described above can be made usingany of the techniques and guidelines for conservative andnon-conservative mutations set forth in U.S. Pat. No. 5,364,934. Theseinclude oligonucleotide-mediated (site-directed) mutagenesis, alaninescanning, and PCR mutagenesis. See also, for example, Table I thereinand the discussion surrounding this table for guidance on selectingamino acids to change, add, or delete.

2. Insertion of Nucleic Acid into Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding the Wntpolypeptide is inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. Many vectors areavailable. The vector components generally include, but are not limitedto, one or more of the following: a signal sequence, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence.

a. Signal sequence component

The Wnt polypeptide useful in hematopoiesis according to the inventionmay be produced recombinantly not only directly, but also as a fusionpolypeptide with a heterologous polypeptide, which is preferably asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe Wnt polypeptide DNA that is inserted into the vector. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processthe native Wnt polypeptide signal sequence, the signal sequence issubstituted by a prokaryotic signal sequence selected, for example, fromthe group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces α-factorleaders, the latter described in U.S. Pat. No. 5,010,182 issued Apr. 23,1991), or acid phosphatase leader, the C. albicans glucoamylase leader(EP 362,179 published Apr. 4, 1990), or the signal described in WO90/13646 published Nov. 15, 1990. In mammalian cell expression thenative signal sequence (e.g., the Wnt polypeptide presequence thatnormally directs secretion of Wnt polypeptide from human cells in vivo)is satisfactory, although other mammalian signal sequences may besuitable, such as signal sequences from other animal Wnt polypeptide,and signal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders, for example, the herpessimplex gD signal.

The DNA for such precursor region is ligated in reading frame to DNAencoding the mature Wnt polypeptide.

b. Origin of replication component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

Most expression vectors are "shuttle" vectors, i.e., they are capable ofreplication in at least one class of organisms but can be transfectedinto another organism for expression. For example. a vector is cloned inE. coli and then the same vector is transfected into yeast or mammaliancells for expression even though it is not capable of replicatingindependently of the host cell chromosome.

DNA may also be amplified by insertion into the host genome. This isreadily accomplished using Bacillus species as hosts, for example, byincluding in the vector a DNA sequence that is complementary to asequence found in Bacillus genomic DNA. Transfection of Bacillus withthis vector results in homologous recombination with the genome andinsertion of Wnt polypeptide DNA. However, the recovery of genomic DNAencoding Wnt polypeptide is more complex than that of an exogenouslyreplicated vector because restriction enzyme digestion is required toexcise the Wnt polypeptide DNA.

c. Selection gene component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This gene encodes a protein necessary forthe survival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate,or tetracycline, (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theWnt polypeptide nucleic acid, such as DHFR or thymidine kinase. Themammalian cell transformantsare placed under selection pressure thatonly the transformants are uniquely adapted to survive by virtue ofhaving taken up the marker. Selection pressure is imposed by culturingthe transformants under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodes Wntpolypeptide. Amplification is the process by which genes in greaterdemand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Increased quantities of Wnt polypeptide aresynthesized from the amplified DNA. Other examples of amplifiable genesinclude metallothionein-I and -II, preferably primate metallothioneingenes, adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub et al., Proc. Nati. Acad. Sci. USA,77: 4216 [1980]. The transformed cells are then exposed to increasedlevels of methotrexate. This leads to the synthesis of multiple copiesof the DHFR gene, and, concomitantly, multiple copies of other DNAcomprising the expression vectors, such as the DNA encoding Wntpolypeptide. This amplification technique can be used with any otherwisesuitable host, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the presenceof endogenous DHFR if, for example, a mutant DHFR gene that is highlyresistant to Mtx is employed (EP 117,060).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) tranrsformed or co-transformed with DNA sequencesencoding Wnt polypeptide, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3'-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39-43 [1979]).The trp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85(1):23-33 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmidpKD1 can beused for transformation of Kluyveromyces yeasts. Bianchi et al., Curr.Genet., 12: 185 [1987]. More recently, an expression system forlarge-scale production of recombinant calf chymosin was reported for K.lactis. Van den Berg, Bio/Technology, 8: 135 [1990]. Stable multi-copyexpression vectors for secretion of mature recombinant human serumalbumin by industrial strains of Kluyveromyces have also been disclosed.Fleer et al., Bio/Technology, 9: 968-975 [1991].

d. Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the Wntpolypeptide nucleic acid. Promoters are untranslated sequences locatedupstream (5') to the start codon of a structural gene (generally withinabout 100 to 1000 bp) that control the transcription and translation ofparticular nucleic acid sequence, such as the Wnt polypeptide nucleicacid sequence, to which they are operably linked. Such promoterstypically fall into two classes, inducible and constitutive. Induciblepromoters are promoters that initiate increased levels of transcriptionfrom DNA under their control in response to some change in cultureconditions, e.g., the presence or absence of a nutrient or a change intemperature. At this time a large number of promoters recognized by avariety of potential host cells are well known. These promoters areoperably linked to Wnt polypeptide-encoding DNA by removing the promoterfrom the source DNA by restriction enzyme,digestion and inserting theisolated promoter sequence into the vector. Both the native Wntpolypeptide promoter sequence and many heterologous promoters may beused to direct amplification and/or expression of the Wnt polypeptideDNA. However, heterologous promoters are preferred, as they generallypermit greater transcription and higher yields of Wnt polypeptide ascompared to the native Wnt polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature,275:617-624 [1978]; Goeddelet al., Nature, 281: 544 [1979]), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8: 4057 [1980]; EP 36,776), and hybrid promoters such as the tacpromoter. deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 [1983].However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding Wnt polypeptide (Siebenlist etal., Cell, 20: 269 [1980]) using linkers or adaptorsto supply anyrequired restriction sites. Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding Wnt polypeptide.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3' end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3' end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J.Biol. Chem., 255: 12073-12080 [1980]) or other glycolytic enzymes (Hesset al., J. Adv. Enzyme Reg., 7: 149 [1968]; Holland, Biochemistry, 17:4900 [1978]), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradativeenzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Wnt polypeptide transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and most preferably Simian Virus 40 (SV40), from heterologousmammalian promoters, e.g., the actin promoter or an immunoglobulinpromoter, from heat-shock promoters, and from the promoter normallyassociated with the Wnt polypeptide sequence, provided such promotersare compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. Fiers et al., Nature 273:113(1978); Mulligan et al.,Science,209: 1422-1427[1980]; Pavlakis et al., Proc. Natl. Acad. Sci.USA 78: 7398-7402 [1981]. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. Greenaway et al., Gene, 18: 355-360 [1982]. A system forexpressing DNA in mammalian hosts using the bovine papilloma virus as avector is disclosed in U.S. Pat. No. 4,419,446. A modification of thissystem is described in U.S. Pat. No. 4,601,978. See also Gray et al.,Nature, 295: 503-508 [1982] on expressing cDNA encoding immuneinterferon in monkey cells; Reyes et al., Nature, 297: 598-601 [1982] onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus; Canaani etal., Proc. Natl. Acad. Sci. USA, 79: 5166-5170 [1982] on expression ofthe human interferon β1 gene in cultured mouse and rabbit cells; andGorman et al, Proc. Natl. Acad. Sci. USA, 79: 6777-6781 [1982] onexpression of bacterial CAT sequences in CV-1 monkey kidney cells,chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, andmouse NIH-3T3 cells using the Rous sarcoma virus long terminal repeat asa promoter.

e. Enhancer element component

Transcription of a DNA encoding the Wnt polypeptide useful inhematopoiesis according to the invention by higher eukaryotes is oftenincreased by inserting an enhancer sequence into the vector. Enhancersare cis-acting elements of DNA, usually about from 10 to 300 bp, thatact on a promoter to increase its transcription. Enhancers arerelatively orientation and position independent, having been found 5'(Laimins et al., Proc. Natl. Acad. Sci. USA 78:464-468 [1981]) and 3'(Lusky et al., Mol. Cell Bio., 3: 1108 [1983]) to the transcriptionunit, within an intron (Banerji et al., Cell, 33: 729 [1983]), as wellas within the coding sequence itself. Osborne et al., Mol. Cell Bio.,4:1293 [1984]). Many enhancersequences are now known from mammaliangenes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5' or 3' to theWnt polypeptide-encoding sequence, but is preferably located at a site5' from the promoter.

f. Transcription termination component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5' and, occasionally 3', untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding Wnt polypeptide.

g. Construction and analysis of vectors

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res., 9: 309 [1981] or by the method of Maxam et al., Methods inEnzymology, 65: 499 [1980].

h. Transient expression vectors

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding Wnt polypeptide. In general, transient expression involvesthe use of an expression vector that is able to replicate efficiently ina host cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high levels of a desiredpolypeptide encoded by the expression vector. Sambrook et al., supra,pp. 16.17-16.22. Transient expression systems, comprising a suitableexpression vector and a host cell, allow for the convenient positiveidentification of polypeptides encoded by cloned DNAs, as well as forthe rapid screening of such polypeptides for desired biological orphysiological properties. Thus, transient expression systems areparticularly useful in the invention for purposes of identifying analogsand variants of Wnt polypeptide that are biologically active Wntpolypeptide.

i. Suitable exemplary vertebrate cell vectors

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of Wnt polypeptide in recombinant vertebrate cell culture aredescribed in Gething et al., Nature 293:620-625 (1981); Manteiet al.,Nature 281: 40-46 [1979]; EP 117,060; and EP 117,058. A particularlyuseful plasmid for mammalian cell culture expression of Wnt polypeptideis pRK5 (EP 307,247) or pSVI6B. WO 91/08291 published Jun 13, 1991.

3. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or highereukaryotecells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting. Strain W3110 is aparticularly preferred host or parent host because it is a common hoststrain for recombinant DNA product fermentations. Preferably, the hostcell should secrete minimal amounts of proteolyticenzymes. For example,strain W3110 may be modified to effect a genetic mutation in the genesencoding proteins, with examples of such hosts including E. coli W3110strain 27C7. The complete genotype of 27C7 is tonAα ptr3phoAΔE15Δ(argF-lac)169ompTΔ degP41kan^(r). Strain 27C7 was deposited onOct. 30, 1991 in the American Type Culture Collection as ATCC No.55,244. Alternatively, the strain of E. coli having mutantperiplasmicprotease disclosed in U.S. Pat. No. 4,946,783 issued Aug. 7,1990 may be employed. Alternatively still, methods of cloning, e.g., PCRor other nucleic acid polymerase reactions, are suitable. In addition toprokaryotes, eukaryotic microbes such as filamentous fungi or yeast aresuitable cloning or expression hosts for Wnt polypeptide-encodingvectors. Saccharomy cescerevisiae, or common baker's yeast, is the mostcommonly used among lower eukaryotic host microorganisms However, anumber of other genera, species, and strains are commonly available anduseful herein, such as Schizosaccharomyces pombe (Beach et al., Nature,290: 140 [1981]; EP 139,383 published May 2, 1985); Kluyveromyces hosts(U.S. Pat. No. 4,943,529; Fleer et al., supra) such as, e.g., K. lactis(MW98-8C, CBS683, CBS4574), K. fragilis (ATCC 12,424), K. bulgaricus(ATCC 16,045), K. wickeramii (ATCC 24,178), K waltii (ATCC 56,500), K.drosophilarum (ATCC 36,906; Van den Berg et al., supra), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol. 28:265-278 (1988));Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case etal., Proc. Natl. Acad. Sci. USA 76:5259-5263 (1979)); Schwanniomycessuch as Schwanniomyces occidentalis (EP 394,538 published Oct. 31,1990); and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium (WO 91/00357 published Jan. 10, 1991), and Aspergillushosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.Commun. 112:284-289 (1983); Tilburn et al., Gene 26:205-221 (1983);Yelton et al., Proc. Natl. Acad. Sci. USA, 81 1470-1474 [1984]) and A.niger. Kelly et al., EMBO J., 4: 475-479 [1985].

Suitable host cells for the expression of glycosylated Wnt polypeptideare derived from multicellular organisms. Such host cells are capable ofcomplex processing and glycosylation activities. In principle, anyhigher eukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture. Examples of invertebrate cells include plant andinsect cells. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts such as Spodopterafrugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori havebeen identified. See, e.g., Luckow et al., Bio/Technology, 6: 47-55[1988]; Miller et al., in Genetic Engineering, Setlow et al., eds., Vol.8 (Plenum Publishing, 1986), pp. 277-279; and Maeda et al., Nature, 315:592-594 [1985]. A variety of viral strains for transfection are publiclyavailable, e.g., the L-1 variant of Autographa california NPV and theBm-5 strain of Bombyx mori NPV, and such viruses may be used as thevirus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens; which has been previously manipulated tocontain the Wnt polypeptide-encoding DNA. During incubation of the plantcell culture with A. tumefaciens, the DNA encoding the Wnt polypeptideis transferred to the plant cell host such that it is transfected, andwill, under appropriate conditions, express the Wnt polypeptide-encodingDNA. In addition, regulatory and signal sequences compatible with plantcells are available, such as the nopaline synthase promoter andpolyadenylation signal sequences. Depicker et al, J. Mol. AppI. Gen., 1:561 [1982]. In addition, DNA segments isolated from the upstream regionof the T-DNA 780 gene are capable of activating or increasingtranscription levels of plant-expressible genes in recombinantDNA-containing plant tissue. EP 321,196 published Jun. 21, 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become a routineprocedure. See, e.g., Tissue Culture, Academic Press, Kruse andPatterson, editors (1973). Examples of useful mammalian host cell linesare monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);human embryonic kidney line (293 or 293 cells subcdoned for growth insuspension culture, Graham et al., J. Gen Virol 36: 59 [1977]); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216[1980]); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Matheret al., Annals N.Y. Acad. Sci.,383: 44-68 [1982]); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors for Wnt polypeptideproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences.

Transfection refers to the taking up of an expression vector by a hostcell whether or not any coding sequences are in fact expressed. Numerousmethods of transfection are known to the ordinarily skilled artisan, forexample, CaPO₄ and electroporation. Successful transfection is generallyrecognized when any indication of the operation of this vector occurswithin the host cell.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomalelement or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., supra, or electroporation is generally used for prokaryotes orother cells that contain substantial cell-wall barriers. Infection withAgrobacterium tumefaciens is used for transformation of certain plantcells, as described by Shaw et al., Gene, 23:315(1983) and WO 89/05859published Jun. 29, 1989. In addition, plants may be transfected usingultrasound treatment as described in WO 91/00358 published Jan. 10,1991.

For mammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham et al., Virology, 52: 456-457 [1973] ispreferred. General aspects of mammalian cell host system transformationshave been described in U.S. Pat. No. 4,399,216 issued Aug. 16, 1983.Transformationsinto yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 [1977] and Hsiao etal., Proc. Natl. Acad. Sci. USA, 76: 3829 [1979]. However, other methodsfor introducing DNA into cells, such as by nuclear microinjection,electroporation, bacterial protoplast fusion with intact cells, orpolycations, e.g., polybrene, polyornithine, etc., may also be used. Forvarious techniques for transforming mammalian cells, see Keown et al.,Methods in Enzymology, 185: 527-537 [1990] and Mansour et al., Nature,336: 348-352 [1988].

4. Culturing the Host Cells

Prokaryotic cells used to produce the Wnt polypeptide useful accordingto the invention are cultured in suitable media as described generallyin Sambrook et al., supra.

The mammalian host cells used to produce the Wnt polypeptide of thisinvention may be cultured in a variety of media. Commercially availablemedia such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM),Sigma), RPMI 1640 (Sigma), and Dulbecco's Modified Eagle's Medium((DMEM), Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham et al., Meth. Enz., 58: 44 [1979],Barnes et al., Anal. Biochem., 102: 255 [1980], U.S. Pat. Nos.4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430;WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used as culture mediafor the host cells. Any of these media may be supplemented as necessarywith hormones and/or other growth factors (such as insulin, transferrin,or epidermal growth factor), salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), traceelements (defined as inorganic compounds usually present at finalconcentrations in the micromolarrange), and glucose oran equivalentenergy source. Any other necessary supplements may also be included atappropriate concentrations that would be known to those skilled in theart. The culture conditions, such as temperature, pH₁ and the like, arethose previously used with the host cell selected for expression, andwill be apparent to the ordinarily skilled artisan.

In general, principles, protocols, and practical techniques formaximizing the productivity of mammalian cell cultures can be found inMammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRLPress, 1991).

The host cells referred to in this disclosure encompass cells in cultureas well as cells that are within a host animal.

5. Detecting Gene Amplificationl Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA (Thomas, Proc Natl.Acad. Sci. USA, 77: 5201-5205 [1980]), dot blotting (DNA analysis), orin situ hybridization, using an appropriately labeled probe, based onthe sequences provided herein. Various labels may be employed, mostcommonly radioisotopes, particularly ³² P. However, other techniques mayalso be employed, such as using biotin-modified nucleotides forintroduction into a polynucleotide. The biotin then serves as the sitefor binding to avidin or antibodies, which may be labeled with a widevariety of labels, such as radionuclides, fluorescers, enzymes, or thelike. Alternatively, antibodies may be employed that can recognizespecific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNAhybrid duplexes or DNA-protein duplexes. The antibodies in turn may belabeled and the assay may be carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemicalstaining techniques,a cell sample is prepared, typically by dehydration and fixation,followed by reaction with labeled antibodies specific for the geneproduct coupled, where the labels are usually visually detectable, suchas enzymatic labels, fluorescent labels, luminescent labels, and thelike. A particularly sensitive staining technique suitable for use inthe present invention is described by Hsu et al., Am. J. Clin. Path.,75: 734-738 [1981].

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared as described herein.

6. Purification of Wnt Polypeptide

Wnt polypeptide may be recovered from the culture medium as a secretedpolypeptide, although it is preferentially recovered from host celllysates. If the Wnt polypeptide is membrane-bound, it can be releasedfrom the cell surface using suitable agents, including enzyms ordetergents (e.g. Triton®X-100), for example, suramin, PMA, heparin,Heparinase I and III, plasmin, n-Octyl-beta-D-glucoside, Pl-specific-and PC-specific-phospholipase C and TNF-alpha.

When Wnt polypeptide is produced in a recombinant cell other than one ofhuman origin, the Wnt polypeptide is completelyfree of proteins orpolypeptides of human origin. However, it is necessary to purify Wntpolypeptide from recombinant cell proteins or polypeptides to obtainpreparations that are substantially homogeneous as to Wnt polypeptide.As a first step, the culture medium or lysate is centrifuged to removeparticulate cell debris. Wnt polypeptide thereafter is purified fromcontaminant soluble proteins and polypeptides, with the followingprocedures being exemplary of suitable purification procedures: byfractionation on an ion-exchange column; ethanol precipitation; reversephase HPLC; chromatography on silica or on a cation-exchange resin suchas DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gelfiltration using, for example, Sephadex®G-75; and protein A Sepharose™columns to remove contaminants such as IgG.

Wnt polypeptide variants in which residues have been deleted, inserted,or substituted are recovered in the same fashion as native sequence Wntpolypeptide, taking account of any substantial changes in propertiesoccasioned by the variation. Immunoaffinity columns such as a rabbitpolyclonal anti-Wnt polypeptide column can be employed to absorb the Wntpolypeptide variant by binding it to at least one remaining immuneepitope.

A protease inhibitorsuch as phenyl methyl sulfonyl fluoride (PMSF) alsomay be useful to inhibit proteolytic degradation during purification,and antibiotics may be included to prevent the growth of adventitiouscontaminants.

7. Covalent Modifications

Covalent modifications of Wnt polypeptide are included within the scopeof this invention. Both native sequence Wnt polypeptide and amino acidsequence variants of the Wnt polypeptide may be covalently modified. Onetype of covalent modification of the Wnt polypeptide is introduced intothe molecule by reacting targeted amino acid residues of the Wntpolypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N- or C-terminal residues ofthe Wnt polypeptide.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleim ides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing a-amino-containingresidues include imidoesterssuch as methyl picolinimidate, pyridoxal phosphate, pyridoxal,chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea,2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed under alkaline conditionsbecause of the high pK_(a) of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas with the arginine epsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵ I or ¹³¹ I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method being suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides(R--N═C═N--R'),where R and R' are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatizationwith bifunctional agents is useful for crosslinkingWntpolypeptideto a water-insoluble support matrix or surface for use in themethod for purifying anti-Wnt polypeptide antibodies, and vice-versa.Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azido-salicylicsalicylic acid, homobifunctionalimidoesters, including disuccinimidylesters such as 3,3'-dithiobis-(succinimidylpropionate), and bifunctionalmaleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents suchas methyl-3-((p-azidophenyl)dithio)propioimidate yield photoactivatableintermediatesthat are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure andMolecularProperties W. H. Freeman & Co., San Francisco, pp. 79-86(1983)), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group.

Another type of covalent modification of the Wnt polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. By altering is meant deletingone or more carbohydrate moieties found in native Wnt polypeptide,and/or adding one or more glycosylation sites that are not present inthe native Wnt polypeptide.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine,galactose, or xylose to a hydroxylamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the Wnt polypeptide is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thenative Wnt polypeptide sequence (for O-linked glycosylation sites). Forease, the Wnt polypeptide amino acid sequence is preferably alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the Wnt polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids. The DNAmutation(s) may be made using methods described above and in U.S. Pat.No. 5,364,934, supra.

Another means of increasing the number of carbohydrate moieties on theWnt polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. These procedures are advantageous in that they do notrequire production of the polypeptide in a host cell that hasglycosylation capabilities for N- or O-linked glycosylation. Dependingon the coupling mode used, the sugar(s) may be attached to (a) arginineand histidine, (b) free carboxyl groups, (c) free sulfhydryl groups suchas those of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 published Sep. 11,1987, and in Aplin et al., CRC Crit. Rev. Biochem. 259-306 (1981).

Removal of carbohydrate moieties present on the Wnt polypeptide may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the polypeptide to the compoundtrifluoromethanesulfonicacid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddin,et al, Arch. Biochem. Biophys. 259:52 (1987) and by Edge et al., Anal.Biochem. 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. EnzymoL 138:350(1987).

Glycosylationat potential glycosylation sites may be prevented by theuse of the compound tunicamycin as described by Duksin et al., J. Biol.Chem. 257:3105 (1982). Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of Wnt polypeptide compriseslinking the Wnt polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol, polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Since it is often difficult to predict in advance the characteristics ofa variant Wnt polypeptide, it will be appreciated that some screening ofthe recovered variant will be needed to select the optimal variant. Achange in the immunological character of the Wnt polypeptide molecule,such as affinity for a given antibody, is also able to be measured by acompetitive-type immunoassay. The Wnt polypeptide variant is assayed forchanges in the ability of the protein to induce cell proliferation inthe assays of Example 2. Other potential modifications of protein orpolypeptide properties such as redox or thermal stability,hydrophobicity, susceptibility to proteolytic degradation, or thetendency to aggregate with carriers or into multimers are assayed bymethods well known in the art.

8. Epitope-Tagged Wnt Polypeptide

This invention encompasses chimeric polypeptides comprising Wntpolypeptide fused to a heterologous polypeptide. A chimeric Wntpolypeptide is one type of Wnt polypeptide variant as defined herein. Inone preferred embodiment, the chimeric polypeptide comprises a fusion ofthe Wnt polypeptide with a tag polypeptidewhich provides an epitope towhich an anti-tag antibody can selectively bind. The epitope tag isgenerally provided at the amino- or carboxyl- terminus of the Wntpolypeptide. Such epitope-tagged forms of the Wnt polypeptide aredesirable as the presence thereof can be detected using a labeledantibody against the tag polypeptide. Also, provision of the epitope tagenables the Wnt polypeptide to be readily purified by affinitypurification using the anti-tag antibody. Affinity purificationtechniques and diagnostic assays involving antibodies are describedlater herein. Tag polypeptides and their respective antibodies are wellknown in the art. Examples include the Herpes Simplex virus glycoproteinD (gD) tag and its antibody (Pennica et al., supra); the his tag, forexample his₆ (Hengen, Trends Biochem. Sci., 20: 285-286 [1995] andPennica et al., J. Biol. Chem., 270: 10915-10922 [1995]); the flu HA tagpolypeptide and its antibody 12CA5 (Field et al., Mol. Cell. Biol., 8:2159-2165 [1988]); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology, 5:3610-3616 [1985]; and Paborsky et al., Protein Engineering, (6): 547-553[1990]). Other tag polypeptides have been disclosed. Examples includethe Flag-peptide (Hopp et al., BioTechnology, 6: 1204-1210 [1988]); theKT3 epitope peptide (Martin et al., Science, 255: 192-194 [1992]); anα-tubulin epitope peptide (Skinneret al., J. Biol. Chem., 266:14163-14166 [1991]); and the T7 gene 10 protein peptide tag.Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-6397[1990]. Once the tag polypeptide has been selected, an antibody theretocan be generated using the techniques disclosed herein.

The general methods suitable for the construction and production ofepitope-tagged Wnt polypeptide are the same as those disclosedhereinabove. Wnt polypeptide-tag polypeptide fusions are mostconveniently constructed by fusing the cDNA sequence encoding the Wntpolypeptide portion in-frame to the tag polypeptide DNA sequence andexpressing the resultant DNA fusion construct in appropriate host cells.Ordinarily, when preparing the Wnt polypeptide-tag polypeptide chimerasof the present invention, nucleic acid encoding the Wnt polypeptide willbe fused at its 3'end to nucleic acid encoding the N-terminus of the tagpolypeptide, however 5' fusions are also possible.

Epitope-tagged Wnt polypeptide can be conveniently purified by affinitychromatography using the anti-tag antibody. The matrix to which theaffinity antibody is attached is most often agarose, but other matricesare available (e.g. controlled pore glass orpoly(styrenedivinyl)benzene). The epitope-taggedwnt polypeptide can beeluted from the affinity column by varying the buffer pH or ionicstrength or adding chaotropic agents, for example.

9. Wnt Polypeptide Immunoadhesins

Chimeras constructed from a receptor sequence linked to an appropriateimmunoglobulin constantdomain sequence (immunoadhesins)are known in theart. Immunoadhesins reported in the literature include fusions of the Tcell receptor* (Gascoigne et al., Proc. Nat. Acad. Sci. USA 84:2936-2940 (1987)); CD4* (Capon et al., Nature 337: 525-531 (1989);Traunecker et al., Nature 339: 68-70 (1989); Zettlmeissl et al., DNACell Biol. USA 9: 347-353 (1990); Byrn et al, Nature 344: 667-670(1990)); L-selectin (homing receptor) ((Watson et al., J. Cell. Biol.110:2221-2229 (1990); Watson et al., Nature 349:164-167(1991)); CD44*(Aruffo et al., Cell61:1303-1313(1990)); CD28* and B7* (Lisley et al.,J. Exp. Med. 173: 721-730 (1991)); CTLA4* (Lisley et al., J. Exp. Med.174: 561-569 (1991)); CD22* (Stamenkovic et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al, Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol 27: 2883-2886(1991); Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); NP receptors(Bennett et al., J. Biol. Chem. 266:23060-23067 (1991)); and IgEreceptor α* (Ridgway et al., J. Cell. Biol. 115:abstr. 1448 (1991)),where the asterisk (*) indicates that the receptor is member of theimmunoglobulin superfamily.

The simplest and most straightforward immunoadhesin design combines thebinding region(s) of the "adhesin" protein with the hinge and Fc regionsof an immunoglobulin heavy chain. Ordinarily, when preparing the Wntpolypeptide-immunoglobulinchimeras of the present invention, nucleicacid encoding Wnt polypeptide will be fused C-terminally to nucleic acidencoding the N-terminus of an immunoglobulin constant domain sequence,however N-terminal fusions are also possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, CH2 and CH3 domains of the constantregion of an immunoglobulin heavy chain. Fusions are also made to theC-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the CH1 of the heavy chain or the corresponding region ofthe light chain.

The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity, secretion or binding characteristics of the Wntpolypeptide-immunoglobulin chimeras.

In some embodiments, the Wnt polypeptide-immunoglobulin chimeras areassembled as monomers, or hetero- or homo-multimers, and particularly asdimers or tetramers, essentially as illustrated in WO 91/08298.

In a preferred embodiment, the Wnt polypeptide sequence is fused to theN-terminus of the C-terminal portion of an antibody (in particularthe Fcdomain), containing the effector functions of an immunoglobulin, e.g.immunoglobulin Gl (IgG1). It is possible to fuse the entire heavy chainconstant region to the Wnt polypeptide sequence. However, morepreferably, a sequence beginning in the hinge region just upstream ofthe papain cleavage site (which defines IgG Fc chemically; residue 216,taking the first residue of heavy chain constant region to be 114, oranalogous sites of other immunoglobulins) is used in the fusion. In aparticularly preferred embodiment, the Wnt polypeptide amino acidsequence is fused to the hinge region, CH2 and CH3, or the CH1, hinge,CH2 and CH3 domains of an IgG1, lgG2, or IgG3 heavy chain. The precisesite at which the fusion is made is not critical, and the optimal sitecan be determined by routine experimentation.

In some embodiments, the Wnt polypeptide-immunoglobulin chimeras areassembled as multimers, and particularly as homo-dimers or -tetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four unit is repeated in the higher molecularweight immunoglobulins; IgM generally exists as a pentamerof basic fourunits held together by disulfide bonds. IgA globulin, and occasionallyIgG globulin, may also exist in multimeric form in serum. In the case ofmultimer, each four unit may be the same or different.

Various exemplary assembled Wnt polypeptide-immunoglobulinchimeraswithin the scope herein are schematically diagrammed below:

(a) AC_(L) -AC_(L) ;

(b) AC_(H) -(AC_(H), AC_(L) -AC_(H), AC_(L) -V_(H) C_(H), or V_(L) C_(L)-AC_(H));

(c) AC_(L) -AC_(H) -(AC_(L) -AC_(H), AC_(L) -V_(H) C_(H), V_(L) C_(L)-AC_(H), or V_(L) C_(L) -V_(H) C_(H));

(d) AC_(L-V) _(H) C_(H) -(AC_(H), or AC_(L) -V_(H) C_(H), or V_(L) C_(L)-AC_(H));

(e) V_(L) C_(L) -AC_(H) -(AC_(L) -V_(H) C_(H), or V_(L) C_(L) -AC_(H));and

(f) (A-Y)_(n),-(V_(L) C_(L) -V_(H) C_(H))₂,

wherein

each A represents identical or different Wnt polypeptide or amino acidsequences;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed asbeing present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the Wnt polypeptide sequence can be inserted betweenimmunoglobulin heavy chain and light chain sequences such that animmunoglobulin comprising a chimeric heavy chain is obtained. In thisembodiment, the Wnt polypeptide sequence is fused to the 3' end of animmunoglobulin heavy chain in each arm of an immunoglobulin, eitherbetween the hinge and the CH2 domain, or between the CH2 and CH3domains. Similar constructs have been reported by Hoogenboom et al, Mol.Immunol., 28:1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to an Wntpolypeptide-immunoglobulin heavy chain fusion polypeptide, or directlyfused to the Wnt polypeptide. In the former case, DNA encoding animmunoglobulin light chain is typically coexpressed with the DNAencoding the Wnt polypeptide-immunoglobulin heavy chain fusion protein.Upon secretion, the hybrid heavy chain and the light chain will becovalently associated to provide an immunoglobulin-like structurecomprising two disulfide-linked immunoglobulin heavy chain-light chainpairs. Methods suitable for the preparation of such structures are, forexample, disclosed in U.S. Pat. No. 4,816,567 issued Mar. 28, 1989.

In a preferred embodiment, the immunoglobulin sequences used in theconstruction of the immunoadhesins of the present invention are from anIgG immunoglobulin heavy chain constant domain. For humanimmunoadhesins, the use of human IgG1 and IgG3 immunoglobulin sequencesis preferred. A major advantage of using IgG1 is that IgG1immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG3 requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG3 hingeis longer and more flexible, so it can accommodate larger adhesindomains that may not fold or function properly when fused to IgG1.Another consideration may be valency; IgG immunoadhesins are bivalenthomodimers, whereas Ig subtypes like IgA and IgM may give rise todimeric or pentameric structures, respectively, of the basic Ighomodimer unit. For immunoadhesins designed for in vivo application, thepharmacokinetic properties and the effector functions specified by theFc region are important as well. Although IgG1, IgG2 and IgG4 all havein vivo half-lives of 21 days, their relative potencies at activatingthe complement system are different. IgG4 does not activate complement,and IgG2 is significantlyweaker at complement activation than IgG1.Moreover, unlike IgG1, IgG2 does not bind to Fc receptors on mononuclearcells or neutrophils. While IgG3 is optimal for complement activation,its in vivo half-life is approximately one third of the other IgGisotypes. Another important consideration for immunoadhesins designed tobe used as human therapeutics is the number of allotypic variants of theparticular isotype. In general, IgG isotypes with fewerserologically-definedallotypes are preferred. For example, IgG1 has onlyfour serologically-defined allotypicsites, two of which (G1m and 2) arelocated in the Fc region; and one of these sites G1m1, isnon-immunogenic. In contrast, there are 12 serological-defined allotypesin IgG3, all of which are in the Fc region; only three of these sites(G3m5, 11 and 21) have one allotype which is nonimmunogenic. Thus, thepotential immunogenicityof a y3 immunoadhesin is greater than that of ay1 immunoadhesin.

With respect to the parental immunoglobulin, a useful joining point isjust upstream of the cysteines of the hinge that form the disulfidebonds between the two heavy chains. In a frequently used design, thecodon for the C-terminal residue of the Wnt polypeptide part of themolecule is placed directly upstream of the codons for the sequenceDKTHTCPPCP (SEQ ID NO:1) of the IgG1 hinge region.

The general methods suitable for the construction and expression ofimmunoadhesins are the same as those disclosed hereinabove with regardto Wnt polypeptide. lmmunoadhesins are most conveniently constructed byfusing the.cDNA sequence encoding the Wnt polypeptide portion in-frameto an Ig cDNA sequence. However, fusion to genomic Ig fragments can alsobe used (see, e.g., Gascoigne et al., Proc. Natl. Acad. Sci. USA,84:2936-2940 (1987); Aruffo et al., Cell 61:1303-1313 (1990);Stamenkovic et al., Cell 66:1133-1144 (1991)). The latter type of fusionrequires the presence of Ig regulatory sequences for expression. cDNAsencoding IgG heavy-chain constant regions can be isolated based onpublished sequence from cDNA libraries derived from spleen or peripheralblood lymphocytes, by hybridization or by polymerase chain reaction(PCR) techniques. The cDNAs encoding the Wnt polypeptide and Ig parts ofthe immunoadhesin are inserted in tandem into a plasmid vector thatdirects efficient expression in the chosen host cells. For expression inmammalian cells, pRK5-based vectors (Schall et al., Cell 61:361-370(1990)) and CDM8-based vectors (Seed, Nature 329:840 (1989)) can beused. The exact junction can be created by removing the extra sequencesbetween the designed junction codons usingoligonucleotide-directeddeletional mutagenesis (Zoller et al., NucleicAcids Res. 10:6487 (1982); Capon et al., Nature 337:525-531 (1989)).Synthetic oligonucleotides can be used, in which each half iscomplementaryto the sequence on either side of the desired junction;ideally, these are 36 to 48-mers. Alternatively, PCR techniques can beused to join the two parts of the molecule in-frame with an appropriatevector.

The choice of host cell line for the expression of the immunoadhesindepends mainly on the expression vector. Another consideration is theamount of protein that is required. Milligram quantities often can beproduced by transient transfections. For example, the adenovirusEIA-transformed 293 human embryonic kidney cell line can be transfectedtransientlywith pRK5-based vectors by a modification of the calciumphosphate method to allow efficient immunoadhesin expression. CDM8-basedvectors can be used to transfect COS cells by the DEAE-dextran method(Aruffo et al., Cell 61:1303-1313 (1990); Zettmeissl et al., DNA CellBiol. US 9:347-353 (1990)). If larger amounts of protein are desired,the immunoadhesin can be expressed after stable transfection of a hostcell line. For example, a pRK5-based vector can be introduced intoChinese hamster ovary (CHO) cells in the presence of an additionalplasmid encoding dihydrofolate reductase (DHFR) and conferringresistance to G418. Clones resistant to G418 can be selected in culture;these clones are grown in the presence of increasing levels of DHFRinhibitor methotrexate; clones are selected, in which the number of genecopies encoding the DHFR and immunoadhesin sequences is co-amplified. Ifthe immunoadhesin contains a hydrophobic leader sequence at itsN-terminus, it is likely to be processed and secreted by the transfectedcells. The expression of immunoadhesins with more complex structures mayrequire uniquely suited host cells; for example, components such aslight chain or J chain may be provided by certain myeloma or hybridomacell hosts (Gascoigne et al., 1987, supra, Martin et al., J. Virol.67:3561-3568 (1993)).

Immunoadhesins can be conveniently purified by affinity chromatography.The suitability of protein A as an affinity ligand depends on thespecies and isotype of the immunoglobulin Fc domain that is used in thechimera. Protein A can be used to purify immunoadhesins that are basedon human y1, y2, or y4 heavy chains (Lindmark et al., J. Immunol. Meth.62:1-13 (1983)). Protein G is recommendedfor all mouse isotypes and forhuman y3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Theconditions for binding an immunoadhesin to the protein A or G affinitycolumn are dictated entirely by the characteristics of the Fc domain;that is, its species and isotype. Generally, when the proper ligand ischosen, efficient binding occurs directly from unconditioned culturefluid. One distinguishing feature of immunoadhesins.is that, for humanγ1 molecules, the binding capacity for protein A is somewhat diminishedrelative to an antibody of the same Fc type. Bound immunoadhesin can beefficiently eluted either at acidic pH (at or above 3.0), or in aneutral pH buffer containing a mildly chaotropic salt. This affinitychromatography step can result in an immunoadhesin preparation thatis >95% pure.

Other methods known in the art can be used in place of, or in additionto, affinity chromatographyon proteinA or G to purify immunoadhesins.Immunoadhesins behave similarly to antibodiesin thiophilicgelchromatography(Hutchens et al., Anal. Biochem. 159:217-226(1986)) andimmobilized metal chelate chromatography (AI-Mashikhi et al., J. DaifySci. 71:1756-1763 (1988)). In contrast to antibodies, however, theirbehavioron ion exchange columns is dictated not only by theirisoelectric points, but also by a charge dipole that may exist in themolecules due to their chimeric nature.

If desired, the immunoadhesins can be made bispecific. Thus, theimmunoadhesins of the present invention may combine a Wnt polypeptideand a domain, such as the extracellular domain, of another cytokinereceptor subunit. Exemplary cytokine receptors from which suchbispecific immunoadhesin molecules can be made include TPO (or mplligand), EPO, G-CSF, IL-4, IL-7, GH, PRL, IL-3, GM-CSF, IL-5, IL-6, LIF,OSM,CNTF and IL-2 receptors. Alternatively, a Wnt polypeptide may becombined with another cytokine, such as those exemplified herein, in thegeneration of a bispecific immunoadhesin. For bispecific molecules,trimeric molecules, composed of a chimeric antibody heavy chain in onearm and a chimeric antibody heavy chain-light chain pair in the otherarm of their antibody-like structure are advantageous, due to ease ofpurification. In contrast to antibody-producing quadromas traditionallyused for the production of bispecific immunoadhesins, which produce amixture of ten tetramers, cells transfected with nucleic acid encodingthe three chains of a trimeric immunoadhesin structure produce a mixtureof only three molecules, and purification of the desired product fromthis mixture is correspondingly easier.

10. Long Half-Life Derivatives of Wnt Polypeptides

Wnt polypeptide functional derivatives for use in the methods of thepresent invention include Wnt-immunoglobulin chimeras (immunoadhesins)and other longer half-life molecules.

Other derivatives of the Wnt polypeptides, which possess a longerhalf-life than the native molecules comprise the Wnt polypeptide or aWnt-immunoglobulin chimera covalently bonded to anonproteinaceouspolymer. The nonproteinaceous polymer ordinarily is ahydrophilic synthetic polymer, i.e., a polymer not otherwise found innature. However, polymers which exist in nature and are produced byrecombinant or in vitro methods are useful, as are polymers which areisolated from native sources. Hydrophilic polyvinyl polymers fall withinthe scope of this invention, e.g. polyvinylalcohol andpolyvinylpyrrolidone. Particularly useful are polyalkylene ethers suchas polyethylene glycol (PEG); polyelkylenes such as polyoxyethylene,polyoxypropylene, and block copolymers of polyoxyethyleneandpolyoxypropylene(Pluronics™); polymethacrylates; carbomers; branched orunbranched polysaccharides which comprise the saccharide monomersD-mannose, D- and L-galactose, fucose, fructose, D-xylose, L-arabinose,D-glucuronic acid, sialic acid, D-galacturonicacid, D-mannuronic acid(e.g. polymannuronic acid, or alginic acid), D-glucosamine,D-galactosamine, D-glucose and neuraminic acid includinghomopolysaccharides and heteropolysaccharides such as lactose,amylopectin, starch, hydroxyethyl starch, amylose, dextrane sulfate,dextran, dextrins, glycogen, or the polysaccharide subunit of acidmucopolysaccharides, e.g. hyaluronic acid; polymers of sugar alcoholssuch as polysorbitol and polymannitol; heparin or heparon. The polymerprior to cross-linking need not be, but preferably is, water soluble,but the final conjugate must be water soluble. In addition, the polymershould not be highly immunogenic in the conjugate form, nor should itpossess viscosity that is incompatible with intravenous infusion orinjection if it is intended to be administered by such routes.

Preferably the polymer contains only a single group which is reactive.This helps to avoid cross-linking of protein molecules. However, it iswithin the scope herein to optimize reaction conditions to reducecross-linking, or to purify the reaction products through gel filtrationor chromatographic sieves to recover substantially homogenousderivatives.

The molecularweight of the polymer may desirably range from about 100 to500,000, and preferably is from about 1,000 to 20,000. The molecularweight chosen will depend upon the nature of the polymer and the degreeof substitution. In general, the greater the hydrophilicity of thepolymer and the greater the degree of substitution, the lower themolecular weight that can be employed. Optimal molecular weights will bedetermined by routine experimentation.

The polymer generally is covalently linked to the Wnt polypeptide or tothe Wnt-immunoglobulin chimera though a multifunctional crosslinkingagent which reacts with the polymer and one or more amino acid or sugarresidues of the Wnt polypeptide or Wnt-immunoglobulin chimera to belinked. However, it is within the scope of the invention to directlycrosslink the polymer by reacting a derivatized polymer with the hybrid,or via versa.

The covalent crosslinking site on the Wnt polypeptide orWnt-immunoglobulin chimera includes the N-terminal amino group andepsilon amino groups found on lysine residues, as well as other amino,imino, carboxyl, sulfhydryl, hydroxyl or other hydrophilic groups. Thepolymer may be covalently bonded directly to the hybrid without the useof a multifunctional (ordinarily bifunctional) crosslinking agent.Covalent binding to amino groups is accomplished by known chemistriesbased upon cyanuric chloride, carbonyl diimidazole, aldehyde reactivegroups (PEG alkoxide plus diethyl acetal of bromoacetaldehyde; PEG plusDMSO and acetic anhydride, or PEG chloride plus the phenoxide of4-hydroxybenzaldehyde, succinimidyl active esters, activateddithiocarbonate PEG, 2,4,5-trichlorophenylcloroformate orP-nitrophenylcloroformate activated PEG). Carboxyl groups arederivatized by coupling PEG-amine using carbodiimide.

Polymers are conjugated to oligosaccharide groups by oxidation usingchemicals, e.g. metaperiodate, or enzymes, e.g. glucose or galactoseoxidase (either of which produces the aldehyde derivative of thecarbohydrate), followed by reaction with hydrazide or amino derivatizedpolymers, in the same fashion as is described by Heitzmann et al.,P.N.A.S. 71:3537-41 (1974) or Bayer et al., Methods in Enzymology2:308-315 (1979), for the labeling of oligosaccharides with biotin oravidin. Further, other chemical or enzymatic methods which have beenused heretofore to link oligosaccharides are particularly advantageousbecause, in general, there are fewer substitutions than amino acid sitesfor derivatization, and the oligosaccharide products thus will be morehomogenous. The oligosaccharide substituents also are optionallymodified by enzyme digestion to remove sugars, e.g. by neuraminidasedigestion, prior to polymer derivatization.

The polymer will bear a group which is directly reactive with an aminoacid side chain, or the N- or C-terminus of the polypeptide linked, orwhich is reactive with the multifunctional cross-linking agent. Ingeneral, polymers bearing such reactive groups are known for thepreparation of immobilized proteins. In order to use such chemistrieshere, one should employ a water soluble polymer otherwise derivatized inthe same fashion as insoluble polymers heretofore employed for proteinimmobilization. Cyanogen bromide activation is a particularly usefulprocedure to employ in crosslinking polysaccharides.

"Water soluble" in reference to the starting polymer means that thepolymer or its reactive intermediate used for conjugation issufficiently water soluble to participate in a derivatization reaction.

"Water soluble" in reference to the polymer conjugate means that theconjugate is soluble in physiological fluids such as blood.

The degree of substitution with such a polymer will vary depending uponthe number of reactive sites on the protein, whether all or a fragmentof the protein is used, whether the protein is a fusion with aheterologous protein (e.g. an Wnt-immunoglobulin chimera), the molecularweight, hydrophilicity and other characteristics of the polymer, and theparticular protein derivatization sites chosen. In general, theconjugate contains about from 1 to 10 polymer molecules, while anyheterologous sequence may be substituted with an essentially unlimitednumber of polymer molecules so long as the desired activity is notsignificantly adversely affected. The optimal degree of cross-linking iseasily determined by an experimental matrix in which the time,temperature and other reaction conditions are varied to change thedegree of substitution, after which the ability of the conjugates tofunction in the desired fashion is determined.

The polymer, e.g. PEG, is cross-linked by a wide variety of methodsknown per se for the covalent modification of proteins withnonproteinaceous polymers such as PEG. Certain of these methods,however, are not preferred for the purposes herein. Cyanuronic chloridechemistry leads to many side reactions, including protein cross-linking.In addition, it may be particularly likely to lead to inactivation ofproteins containing sulfhydryl groups. Carbonyl diimidazole chemistry(Beauchamp et al., Anal Biochem. 131:25-33 (1983)) requires high pH(>8.5), which can inactivate proteins. Moreover, since the "activatedPEG" intermediate can react with water, a very large molar excess of"activatedPEG" over protein is required. The high concentrationsof PEGrequired for the carbonyl diimidazole chemistry also led to problems inpurification, as both gel filtration chromatography and hydrophilicinteraction chromatography are adversely affected. In addition, the highconcentrationsof "activated PEG" may precipitate protein, a problem thatperse has been noted previously (Davis, U.S. Pat. No. 4,179,337). On theother hand, aldehyde chemistry (Royer, U.S. Pat. No. 4,002,531) is moreefficient since it requires only a 40-fold molar excess of PEG and a 1-2hr incubation. However, the manganese dioxide suggested by Royer forpreparation of the PEG aldehyde is problematic "because of thepronounced tendency of PEG to form complexes with metal-based oxidizingagents" (Harris et al., J. Polym. Sci. Polym. Chem. Ed. 22:341-52(1984)). The use of a Moffatt oxidation, utilizing DMSO and aceticanhydride, obviates this problem. In addition, the sodium borohydridesuggested by Royer must be used at high pH and has a significanttendencyto reduce disulfide bonds. In contrast, sodium cyanoborohydride, whichis effective at neutral pH and has very little tendency to reducedisulfide bonds is preferred.

Functionalized PEG polymers to modify the Wnt polypeptide orWnt-immunoglobulin chimeras of the present invention are available fromShearwater Polymers, Inc. (Huntsville, Ala.). Such commerciallyavailable PEG derivatives include, but are not limited to, amino-PEG,PEG amino acid esters, PEG-hydrazide, PEG-thiol, PEG-succinate,carboxymethylated PEG, PEG-propionic acid, PEG amino acids, PEGsuccinimidyl succinate, PEG succinimidyl propionate, succinimidyl esterof carboxymethylated PEG, succinimidyl carbonate of PEG, succinimidylesters of amino acid PEGs, PEG-oxycarbonylimidazole,PEG-nitrophenylcarbonate, PEG tresylate, PEG-glycidyl ether, PEG-aldehyde, PEGvinylsulfone, PEG-maleimide, PEG-orthopyridyl-disulfide,heterofunctional PEGs, PEG vinyl derivatives, PEG silanes, and PEGphospholides. The reaction conditions for coupling these PEG derivativeswill vary depending on the protein, the desired degree of PEGylation,and the PEG derivative utilized. Some factors involved in the choice ofPEG derivatives include: the desired point of attachment (lysine orcysteine), hydrolytic stability and reactivity of the derivatives,stability, toxicity and antigenicity of the linkage, suitability foranalysis, etc. Specific instructions for the use of any particularderivative are available from the manufacturer.

The long half-life conjugates of this invention are separated from theunreacted starting materials by gel filtration. Heterologous species ofthe conjugates are purified from one another in the same fashion. Thepolymer also may be water-insoluble, as a hydrophilic gel.

The conjugates may also be purified by ion-exchange chromatography. Thechemistry of many of the electrophilically activated PEG's results in areduction of amino group charge of the PEGylated product. Thus, highresolution ion exchange chromatography can be used to separate the freeand conjugated proteins, and to resolve species with different levels ofPEGylation. In fact, the resolution of different species (e.g.containing one or two PEG residues) is also possible due to thedifference in the ionic properties of the unreacted amino acids.

B. Therapeutic Uses for the Wnt Polypeptide

The Wnt polypeptide and Wnt polypeptide gene are believed to findtherapeutic use for administration to a mammal in the treatment ofdiseases or disorders characterized by a decrease in hematopoieticcells. Examples of these diseases or disorders include: anemia(including macrocytic and aplastic anemia); thrombocytopenia;hypoplasia; disseminated intravascular coagulation (DIC);myelodysplasia; immune (autoimmune) thrombocytopenic purpura (ITP); andHIV induced ITP. Additionally, these Wnt polypeptide molecules may beuseful in treating patients having suffered a hemorrhage. Wntpolypeptide and Wnt polypeptide gene which lead to an increase inhematopoietic cell proliferation may also be used to enhancerepopulation of mature blood cell lineages in cells having undergonechemo- or radiation therapy or bone marrow transplantation therapy.Generally, these molecules are expected to lead to an enhancement of theproliferation, differentiation and/or maintenance of primitivehematopoietic cells.

The Wnt polypeptide may be administered alone or in combination with oneor more cytokines, including growth factors or antibodies in theabove-identified clinical situations. This may facilitate an effectivelowering of the dose of Wnt polypeptide. Suitable dosages for suchadditional molecules will be discussed below.

In gene therapy applications, genes are introduced into cells in orderto achieve in vivo synthesis of a therapeutically effective geneticproduct, for example for replacement of a defective gene. "Gene therapy"includes both conventional gene therapy where a lasting effect isachieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA, 83:4143-4146 (1986)). The oligonucleotides can bemodified to enhance their uptake, e.g., by substituting their negativelycharged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11:205-210 (1993)).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262:4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87:3410-3414 (1990). For review of the currently knowngene marking and gene therapy protocols see Anderson et al, Science256:808-813 (1992).

For therapeutic applications, the Wnt polypeptide useful according tothe invention are administered to a mammal, preferably a human, in aphysiologically acceptable dosage form, including those that may beadministered to a human intravenously as a bolus or by continuousinfusion over a period of time. Alternative routes of administrationinclude intramuscular, intraperitoneal, intra-cerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. The Wnt polypeptides also are suitablyadministered by intratumoral, peritumoral, intralesional, orperilesional routes or to the lymph, to exert local as well as systemictherapeutic effects.

Such dosage forms encompass physiologically acceptable carriers that areinherently non-toxic and non-therapeutic. Examples of such carriersinclude ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts, orelectrolytes such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, and PEG. Carriers for topical or gel-based forms of Wntpolypeptides include polysaccharidessuch as sodiumcarboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, PEG, andwood wax alcohols. For all administrations, conventional depot forms aresuitably used. Such forms include, for example, microcapsules,nano-capsules, liposomes, plasters, inhalation forms, nose sprays,sublingual tablets, and sustained-release preparations. The Wntpolypeptide will typically be formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml.

Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing the Wntpolypeptide, which matrices are in the form of shaped articles, e.g.films, or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et aL, supra and Langer, supra, orpoly(vinylalcohol), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate (Sidman et al, supra),non-degradable ethylene-vinyl acetate (Langer et al., supra), degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolicacid copolymerand leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated Wnt polypeptidesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S-S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

Sustained-release Wnt polypeptide compositions also include liposomallyentrapped polypeptides. Liposomes containing the Wnt polypeptide areprepared by methods known in the art, such as described in Eppstein etal., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and4,544,545. Ordinarily, the liposomes are the small (about 200-800Angstroms) unilamelar type in which the lipid content is greater thanabout 30 mol.% cholesterol, the selected proportion being adjusted forthe optimal Wnt polypeptide therapy. Liposomes with enhanced circulationtime are disclosed in U.S. Pat. No. 5,013,556.

For the prevention or treatment of disease, the appropriate dosage ofWnt polypeptide will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theantibodies are administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to the Wntpolypeptide, and the discretion of the attending physician. The Wntpolypeptideis suitably administeredto the patient at one time or over aseries of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg of Wnt polypeptide is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to 100 μg/kg (e.g. 1-50 μg/kg) ormore, depending on the factors mentioned above. For example, the dosemay be the same as that for other cytokines such as G-CSF, GM-CSF andEPO. For repeated administrations over several days or longer, dependingon the condition, the treatment is sustained until a desired suppressionof disease symptoms occurs. However, other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques and assays.

When one or more cytokines are co-administered with the Wnt polypeptide,lesser doses of the Wnt polypeptide may be employed. Suitable doses of acytokine are from about 1 μg/kg to about 15 mg/kg of cytokine. A typicaldaily dosage of the cytokine might range from about 1 μg/kg to 100 μg/kg(e.g. 1-50 μg/kg) or more. For example, the dose may be the same as thatfor other cytokines such as G-CSF, GM-CSF and EPO. The cytokine(s) maybe administered prior to, simultaneously with, or followingadministration of the Wnt polypeptide. The cytokine(s) and Wntpolypeptide may be combined to form a pharmaceutically composition forsimultaneous administration to the mammal. In certain embodiments, theamounts of Wnt polypeptide and cytokine are such that a synergisticrepopulation of blood cells (or synergistic increase in proliferationand/or differentiation of hematopoietic cells) occurs in the mammal uponadministration of the Wnt polypeptide and cytokine thereto. In otherwords, the coordinated action of the two or more agents (i.e. the Wntpolypeptide and cytokine(s)) with respect to repopulation of blood cells(or proliferation/differentiation of hematopoietic cells) is greaterthan the sum of the individual effects of these molecules.

Therapeutic formulations of Wnt polypeptide are prepared for storage bymixing Wnt polypeptide having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed.,(1980)), in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counter-ions such as sodium; and/or non-ionic surfactantssuch as Tween®, Pluronics™ or polyethylene glycol (PEG).

The Wnt polypeptide also may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsulesandpoly-(methylmethacylate)microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles, and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

Wnt polypeptide to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution. Wntpolypeptide ordinarily will be stored in lyophilized form or insolution. Therapeutic Wnt polypeptide compositions generally are placedinto a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

When applied topically, the Wnt polypeptide is suitably combined withother ingredients, such as carriers and/or adjuvants. There are nolimitations on the nature of such other ingredients, except that theymust be physiologically acceptable and efficacious for their intendedadministration, and cannot degrade the activity of the activeingredients of the composition. Examples of suitable vehicles includeointments, creams, gels, or suspensions, with or without purifiedcollagen. The compositions also may be impregnated into transdermalpatches, plasters, and bandages, preferably in liquid or semi-liquidform.

For obtaining a gel formulation, the Wnt polypeptide formulated in aliquid composition may be mixed with an effective amount of awater-soluble polysaccharide or synthetic polymer such as PEG to form agel of the properviscosityto be applied topically. The polysaccharidethat may be used includes, for example, cellulose derivatives such asetherified cellulose derivatives, including alkyl celluloses,hydroxyalkyl celluloses, and alkylhydroxyalkyl celluloses, for example,methylcellulose, hydroxyethyl cellulose, carboxymethyl cellulose,hydroxypropyl methylcellulose, and hydroxypropyl cellulose; starch andfractionated starch; agar; alginic acid and alginates; gum arabic;pullullan; agarose; carrageenan; dextrans; dextrins; fructans; inulin;mannans; xylans; arabinans; chitosans; glycogens; glucans; and syntheticbiopolymers; as well as gums such as xanthan gum; guar gum; locust beangum; gum arabic; tragacanth gum; and karaya gum; and derivatives andmixtures thereof. The preferred gelling agent herein is one that isinert to biological systems, nontoxic, simple to prepare, and not toorunny or viscous, and will not destabilize the Wnt polypeptide heldwithin it.

Preferably the polysaccharide is an etherified cellulose derivative,more preferably one that is well defined, purified, and listed in USP,e.g., methylcellulose and the hydroxyalkyl cellulose derivatives, suchas hydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropylmethylcellulose. Most preferred herein is methylcellulose.

The polyethylene glycol useful for gelling is typically a mixture of lowand high molecular weight PEGs to obtain the proper viscosity. Forexample, a mixture of a PEG of molecular weight 400-600 with one ofmolecularweight 1500 would be effective for this purpose when mixed inthe proper ratio to obtain a paste.

The term "water soluble" as applied to the polysaccharides and PEGs ismeant to include colloidal solutions and dispersions. In general, thesolubility of the cellulose derivatives is determined by the degree ofsubstitution of ether groups, and the stabilizing derivatives usefulherein should have a sufficient quantity of such ether groups peranhydroglucose unit in the cellulose chain to render the derivativeswater soluble. A degree of ether substitution of at least 0.35 ethergroups per anhydroglucose unit is generally sufficient. Additionally,the cellulose derivatives may be in the form of alkali metal salts, forexample, the Li, Na, K, or Cs salts.

If methylcellulose is employed in the gel, preferably it comprises about2-5%, more preferably about 3%, of the gel and the Wnt polypeptide ispresent in an amount of about 300-1000 mg per ml of gel.

An effective amount of Wnt polypeptide to be employed therapeuticallywill depend, for example, upon the therapeutic objectives, the route ofadministration, and the condition of the patient. Accordingly, it willbe necessary for the therapist to titer the dosage and modify the routeof administration as required to obtain the optimal therapeutic effect.Typically, the clinician will administerthe Wnt polypeptide until adosage is reached that achieves the desired effect. A typical dailydosage for systemic treatment might range from about 1 μg/kg to up to 10mg/kg or more, depending on the factors mentioned above. As analternative general proposition, the Wnt polypeptide receptor isformulated and delivered to the target site or tissue at a dosagecapable of establishing in the tissue a Wnt polypeptide level greaterthan about 0.1 ng/cc up to a maximum dose that is efficacious but notunduly toxic. This intra-tissue concentration should be maintained ifpossible by the administration regime, including by continuous infusion,sustained release, topical application, or injection at empiricallydetermined frequencies. The progress of this therapy is easily monitoredby conventional assays.

C. Non-Therapeutic Uses for the Wnt Polypeptide

Wnt nucleic acid is useful for the preparation of Wnt polypeptide byrecombinant techniques exemplified herein which can then be used forproduction of anti-Wnt antibodies having various utilities describedbelow.

The Wnt polypeptide (polypeptide or nucleic acid) can be used to induceproliferation and/or differentiation of cells in vitro. In particular,it is contemplated that this molecule may be used to induceproliferation of stem cell/progenitorcell populations (e.g. flASK cellpopulations obtained as described in Example 2 below). These cells whichare to be grown ex vivo may simultaneously be exposed to other knowngrowth factors or cytokines, such as those described herein. Thisresults in proliferation, differentiation and/or maintenance of thecells.

In yet another aspect of the invention, the Wnt polypeptide may be usedfor affinity purification of Wnt receptor. Briefly, this techniqueinvolves: (a) contacting a source of Wnt receptor with an immobilizedWnt polypeptide under conditions whereby the Wnt receptor to be purifiedis selectively adsorbed onto the immobilized receptor; (b) washing theimmobilized Wnt polypeptide and its support to remove non-adsorbedmaterial; and (c) eluting the Wnt receptor molecules from theimmobilizedWnt polypeptideto which they are adsorbed with an elutionbuffer. In an embodiment of affinity purification, Wnt polypeptide iscovalently attaching to an inert and porous matrix (e.g., agarosereacted with cyanogen bromide). Preferred is a Wnt polypeptideimmunoadhesin immobilized on a protein A column. A solution containingWnt receptor is then passed through the chromatographic material. TheWnt receptor adsorbs to the column and is subsequently released bychanging the elution conditions (e.g. by changing pH or ionic strength).

The Wnt polypeptide may be used for competitive screening of potentialagonists or antagonists for binding to the cell surface receptors. Suchagonists or antagonists may constitute potential therapeutics.

A preferred technique for identifying molecules which bind to the Wntpolypeptide utilizes a chimeric polypeptide (e.g., epitope tagged Wntpolypeptide or Wnt polypeptide immunoadhesin) attached to a solid phase,such as the well of an assay plate. Binding of molecules which areoptionally labelled (e.g., radiolabelled) to the immobilized receptorcan be evaluated.

The Wnt polypeptides are also useful as molecular weight markers. To usea Wnt polypeptide as a molecular weight marker, gel filtrationchromatography or SDS-PAGE, for example, will be used to separateprotein(s) for which it is desired to determine their molecularweight(s) in substantiallythe normal way. The Wnt polypeptide and othermolecularweight markers will be used as standards to provide a range ofmolecular weights. For example, phosphorylase b (mw=97,400), bovineserum albumin (mw=68,000), ovalbumin (mw=46,000), a Wnt polypeptide(e.g., mw=38,000-42,000 depending on the coding sequence as described byGavin et al., supra), trypsin inhibitor (mw=20,100), and lysozyme(mw=14,400) can be used as mw markers. The other molecularweight markersmentioned here can be purchased commercially from Amersham Corporation,Arlington Heights, Ill. The molecular weight markers are generallylabeled to facilitate detection thereof. For example, the markers may bebiotinylated and following separation can be incubated withstreptavidin-horseradishperoxidase so that the various markers can bedetected by light detection.

The purified Wnt polypeptide, and the nucleic acid encoding it, may alsobe sold as reagents for mechanism studies of Wnt polypeptide and itsreceptor, to study the role of the Wnt polypeptide and Wnt receptor innormal growth and development, as well as abnormal growth anddevelopment, e.g., in malignancies, or in diseases or disorders.

D. Wnt Polypeptide Antibody Preparation

1. Polyclonal antibodies

Potential therapeutic applications for anti-Wnt antibodies, inparticular neutralizing antibodies, include the treatment of disorders,stem cell tumors and other tumors at sites of Wnt expression, includingthose tumors characterized by overexpressions of Wnts.

Polyclonal antibodies are generally raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹ N═C═NR,where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining 1 mg or 1 μg of the peptide or conjugate (forrabbits or mice, respectively) with 3 volumes of Freund's completeadjuvant and injecting the solution intradermally at multiple sites. Onemonth later the animals are boosted with 1/5 to 1/10 the original amountof peptide or conjugate in Freund's complete adjuvant by subcutaneousinjection at multiple sites. Seven to 14 days later the animals are bledand the serum is assayed for antibody titer. Animals are boosted untilthe titer plateaus. Preferably, the animal is boosted with the conjugateof the same antigen, but conjugated to a different protein and/orthrough a different cross-linking reagent. Conjugates also can be madein recombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

2. Monoclonal antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier"monoclonal" indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature 256:495 (1975), or maybe made by recombinant DNA methods (Cabilly et al., supra).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp.59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-produdngcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133: 3001 [1984]); Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 (MarcelDekker, Inc., New York, [1987]).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubdloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subdlones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose®, hydroxylapatte chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5: 256-262 [1993] and Pluckthun, Immunol. Revs., 130: 151-188 [1992].

In a further embodiment, antibodies or antibody fragments can beisolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature 348: 552-554 [1990]. Clackson etal., Nature, 352: 624-628 [1991] and Marks et al., J. Mol. Biol., 222:581-597 [1991] describe the isolation of murine and human antibodies,respectively, using phage libraries. Subsequent publications describethe production of high affinity (nM range) human antibodies by chainshuffling (Mark et al., Bio/Technology, 10: 779-783 [1992]), as well ascombinatorial infection and in vivo recombinationas a strategy forconstructing very large phage libraries (Waterhouse et al., Nuc. Acids.Res., 21: 2265-2266 [1993]). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (Cabilly et al., supra; Morrison, etal., Proc. Nat. Acad. Sci. USA 81: 6851 [1984]), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

3. Humanized and human antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as "import" residues, whichare typically taken from an "import" variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 [1988]; Verhoeyen et al., Science, 239: 1534-1536 [1988]), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such "humanized" antibodiesare chimeric antibodies (Cabilly et al., supra), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called "best-fit" method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151: 2296 [1993]; Chothia et al., J. Mol. Biol., 196: 901[1987]). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89: 4285 [1992]; Presta et al., J. Immnol., 151: 2623 [1993]).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliarto those skilled in the art. Computerprograms are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i. e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the consensus and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possibleto producetransgenicanimals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chainjoining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551[1993]; Jakobovits et al., Nature, 362: 255-258 [1993]; Bvuggermann etal., Year in Immuno., 7: 33 [1993]. Human antibodies can also beproduced in phage- display libraries (Hoogenboom et al., J. Mol. Biol.,227: 381 [1992]; Marks et al., J. Mol. Biol., 222: 581 [1991]).

4. Bispecific antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different antigens. BsAbs can be used astumor targeting or imaging agents and can be used to target enzymes ortoxins to a cell possessing the Wnt polypeptide. Such antibodies can bederived from full length antibodies or antibody fragments (e.g. F(ab')₂bispecific antibodies). In accordance with the present invention, theBsAb may possess one arm which binds the Wnt polypeptide and another armwhich binds to a cytokine or another cytokine receptor (or a subunitthereof) such as the receptors for TPO, EPO, G-CSF, IL-4, IL-7, GH, PRL;the α or β subunits of the IL-3, GM-CSF, IL-5, IL-6, LIF, OSM and CNTFreceptors; or the α, β or γ subunits of the IL-2 receptor complex.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Milstein et al.,Nature 305:537-540 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, published May 13, 1993, and inTraunecker et al., EMBO J., 10: 3655-3659 [1991].

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2, and CH3 regions. Itis preferred to have the first heavy-chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are co-transfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In a preferred embodiment of this approach,.the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690 published Mar. 3,1994. For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121: 210 [1986].

Bispecific antibodies include cross-linked or "heteroconjugate"antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/20373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. The following techniques canalso be used for the production of bivalent antibody fragments which arenot necessarily bispecific. According to these techniques, Fab'-SHfragments can be recovered from E. coli, which can be chemically coupledto form bivalent antibodies. Shalaby et al., J. Exp. Med., 175: 217-225[1992] describe the production of a fully humanized BsAb F(ab')₂molecule. Each Fab' fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the BsAb. TheBsAb thus formed was able to bind to cells overexpressingthe HER2receptor and normal human T cells, as well as triggerthe lytic activityof human cytotoxic lymphocytes against human breast tumor targets. Seealso Rodrigues et al., Int. J. Cancers (Suppl.), 7: 45-50 [1992].Various techniques for making and isolating bivalent antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bivalent heterodimers have been produced using leucine zippers.Kostelnyet al., J. Immunol., 148(5): 1547-1553 [1992]. The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab'portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. The "diabody" technologydescribed by Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448[1993] has provided an alternative mechanism for making BsAb fragments.The fragments comprise a heavy-chainvariable domain (V_(H)) connected toa light-chain variable domain (V_(L)) by a linker which is too short toallow pairing between the two domains on the same chain. Accordingly,the V_(H) and V_(L) domains of one fragment are forced to pair with thecomplementary V_(L) and V_(H) domains of another fragment, therebyforming two antigen-binding sites. Another strategy for making BsAbfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol. 152:5368 (1994).

E. Articles of Manufacture

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of the conditionsdescribed above is provided. The article of manufacture comprises acontainer and a label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is effective for treating the condition andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The active agent in the composition is theWnt polypeptide. The label on, or associated with, the containerindicates that the composition is used for treating the condition ofchoice. The article of manufacture may further comprise a secondcontainer holding a cytokine for co-administration with the Wntpolypeptide. Further container(s) may be provided with the article ofmanufacturewhich may hold, for example, a pharmaceutically-acceptablebuffer, such as phosphate-buffered saline, Ringer's solution or dextrosesolution. The article of manufacture may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

F. Non-Therapeutic Uses for Antibodies to Wnts

Wnt polypeptide antibodies are also useful as affinity purificationagents. In this process, the antibodies against Wnts are immobilized ona suitable support, such a Sephadex® resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the Wnts to be purified, andthereafter the support is washed with a suitable solvent that willremove substantiallyall the material in the sample except the Wnts,which is bound to the immobilized antibody. Finally, the support iswashed with another suitable solvent, such as glycine buffer, pH 5.0,that will release the Wnt polypeptide from the antibody.

Wnts antibodies may also be useful in diagnostic assays for Wntpolypeptide, e.g., detecting its expression in specific cells, tissues,or serum. For diagnostic applications, antibodies typically will belabeled with a detectable moiety. The detectable moiety can be any onewhich is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; radioactive isotopic labels, such as, e.g., ¹²⁵I, ³² P, ¹⁴ C, or ³ H; or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the polypeptidevariant to the detectable moiety may be employed, including thosemethods described by Hunter et al., Nature, 144: 495-496 [1962]; Davidet al., Biochemistry, 13: 1014 [1974]; Pain et al., J. Immunol. Meth,40: 219 [1981]; and Nygren, J. Histochem. and Cytochem., 30: 407 [1982].

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample analyte for binding with a limited amountof antibody. The amount of Wnt polypeptide in the test sample isinversely proportional to the amount of standard that becomes bound tothe antibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition, so that the standard and analyte that are boundto the antibodies may conveniently be separated from the standard andanalyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insolublethree-part complex. See, e.g., U.S. Pat. No. 4,376,1 10. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulinantibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

III. Experimental

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

The disclosures of all publications, patents and patent applicationscited herein, whether supra or infra, are hereby incorporated byreference in their entirety.

EXAMPLE 1 Screening of Wnt Genes in Cells and Cell Lines

Both hematopoieticstem cell populationsand stromal cell lineswhichsupport hematopoietic stem/progenitor cell growth were surveyed for Wntexpression by RT-PCR analysis. For these experiments, fetal liver andbone marrow hematopoietic stem/progenitor cell populations were preparedessentially as described (see, e.g., Example 2A of U.S. Ser. No.08/222,299, corresponding PCT/US95/03718; and Zeigler et al., Blood, 84:2422-2430 [1994].

Briefly, day 14-15 fetal livers were made into a single cell suspensionand AA4⁺ cells were positively selected by immunoadherentpanning usingthe AA4⁺ antibodies purified from hybridoma supernatents as described byZeigleret al., supra. Sca⁺ c-kit⁺ dual positive cells were recoveredfrom the AA4⁺ cell population by flow cytometric sorting using the Ly6A/E phycoerythrin conjugate (Pharmingan, San Diego, Calif.) to recoverSca⁺ cells and using fluorescein conjugated antibodies to c-kit (alsofrom Pharmingan). Lin^(lo) Sca⁺ bone marrow cells were recovered bymagnetic bead depletion (Dynal, Inc., Great Neck, N.J.; Ploemacheretal., Blood, 74: 2755-2763 [1989]) of lineage-antigen expressing cellsfrom total bone marrow and selection of Lin^(lo) Sca⁺ cells by flowcytometric sorting using Lin cocktail antibodiesfrom Caltag (SouthFrancisco, Calif.) as described (see, Example 2A of U.S. Ser. No.08/222,299, corresponding PCT/US95/03718; and Zeigler et al., supra.

For the RT-PCR analysis of these cell subpopulations, PCRs were carriedout with Taq polymerase (Cetus) on AA4⁺ Sca⁺ cDNA or a 7-4 cell linecDNA library using each sense primer (LL1-7) with the Wnt3 antisenseprimer (sense primers: LL1 5' CAA GAG TGC AAA TGC CAC GGG ATG TCC GGCTCC TGC 3'(SEQ ID NO: 2) LL2 5' CAA GAG TGC AAA TGC CAC GGG GTG TCC GGCTCC TGC 3'(SEQ ID NO: 3); LL3 5' CTC AAG TGC AAA TGC CAC GGG CTA TCT GGCAGC TAGT 3'(SEQ ID NO: 4); LL 4 5' GTG GAG TGC AAG TGC CAC GGG GTG TCCGGC TCC TGC 3' (SEQ ID NO: 5); LL5 5' GTA GCC TGT AAG TGC CAT GGA GTGTCT GGC TCC TGT 3' (SEQ ID NO: 6); LL6 5' ACC GGG TGT AAG TGC CAT GGGCTT TCG GGT TCC TGC 3' (SEQ ID NO: 7); LL7 5' CTG GAG TGT AAG TGC CATGGT GTG TCA GGC TCC TGT 3' (SEQ ID NO: 8); antisense primer "Wnt3" 5'GCC (C/G)CG GCC (G/A)CA (G/A)CA CAT3' (SEQ ID NO: 9)). Additional PCRswere performed with the primer Wnt1a (5'(G/C)TG GA(A/G) TG(C/T) AA(A/G)TG(C/T) CAT 3' (SEQ ID NO: 10) and Wnt2a5' (A/G)CA (A/G)CA CCA (A/G)TG(A/G)AA3' (SEQ ID NO: 11). PCR products were cloned as a blunt-endedfragments into Smal-linearized pGEM®7 (Promega) and screened for Wntsequences by hybridization with the oligonucleotides Liem1 5' GAC CTGGTG TAC 3' (SEQ ID NO: 12) or Liem2 5' TG(T/C) TG(T/C) GGC CG(G/C) GGC3' (SEQ ID NO: 13). This analysis was confirmed using the followingspecific primers for Wnt-5a and Wnt-10b, using Wnt-3a as a negativecontrol: Wnt-3a (wn3a.2,5' CAG CCC AGG CGT CCG CGC TC3' (SEQ ID NO: 14);wn3a.3, 5'GGA ATG AAC CCT GCT CCC GT 3' (SEQ ID NO: 15), Wnt-5a(wn5a1050, 5' CGC GCC CCA AGG ACC TGC CTC G 3' (SEQ ID NO: 16);wn5ar1499, 5' GCG AGC CAG TGC TCT CGT TGC G 3' (SEQ ID NO: 17); Wnt-10b(W10.1, 5' AAA CCT GAA GCG GAA GTG CAA ATG C 3' (SEQ ID NO: 18); w10.3,5' GCT CAC CTT CAT TTA CAC ACA TTG A 3' (SEQ ID NO: 19).

These experiments detected only Wnt-5a and Wnt-10b in fetal liver AA4⁺Sca⁺ cells. Similar experiments were performed on a fetal liver stromalcell line, 7-4 (prepared as described in co-pending and coassigned U.S.patent application Ser. No. 08/222,299; corresponding PCT/US95/03718(WO95/27062); and Zeigleret al., supra), where expression of only Wnt-5awas detected. Thus highly enriched stem/progenitorcells and stromalcells that support their expansion were found to express Wnts.

These observations were extended by RT-PCR on mrRNAs from the highlyenriched fetal liver stem cell population AA4⁺ Sca⁺ kit⁺ ("flASK" cells,prepared as described in co-pending and coassigned U.S. patentapplication Ser. No. 08/222,299; corresponding PCT/US95/03718(WO95/27062); and Zeigleret al., supra, using the antibody reagentsdescribed above). Wnt-5a, and Wnt-10b, but not Wnt-3a, were detected inthe flASK cells. Moreover, the lack of Wnt-3a expression in flASK cellsdemonstrated that, at the level of detection of the RT-PCR assays,hematopoietic stem cells express only a subset of the possible Wntgenes.

In additional experiments, Wnt-5a and Wnt-10b mRNAs were shown to beexpressed in the stem cell populationsAA4⁺, AA4⁺ Sca⁺ flk2⁺ and AA4⁺Sca⁺ flk2(Zeigleret al.,supra). Importantly, the expression of these WntmRNAs in three different hematopoietic stem/progenitor cell subsets,each purified independently by virtue of c-kit or flk2 expression, andall three cell subsets capable of long term engraftment of lethallyirradiated animals, strongly suggested a role for these ligands in thelocal microenvironmentof the hematopoietic stem/progenitor cell. Theexpression of Wnt-5a in a fetal liver stromal cell line, 7-4, describedabove, and of Wnt-5a and Wnt-10b mRNAs in more mature (AA4⁻) fetal liverhematopoietic cells further suggested (i) that much of the fetal liverhematopoietic microenvironment, as well as hematopoietic stem/progenitorcells, could potentially serve as a source of Wnts and (ii) that theexpression of Wnt on these cells could allow for Wnt-mediated paracrineor autocrine regulation of the differentiation of these cells.

EXAMPLE 2 Analysis of Wnts in Hematopoiesis

A. Expansion of Hematopoietic Stem/Progenitor Cells Stimulated byConditioned Media From Wnt-Transfected Cells

An in vitro stroma-free suspension culture system was developed to studythe function of Wnts on highly enriched hematopoieticstem/progenitorcell populations prepared as described in Example 1. For theseexperiments, suspension culture of the enriched sorted cells was carriedout in 24 well Costar dishes with 5000 cells/well seeded into 0.5 ml ofHSC media and cultured at 37° C. with 5% CO₂. HSC media contained 50%F12/50% low glucose DMEM, 10% heat-treated fetal bovine serum (Hyclone),1 mM Glutamine, and murine kit ligand (KL) as indicated (R&D Systems).Conditioned media from 293 cells transfected with cloned Wnt genes wasadded at the time of plating. Specifically, Wnt-5a cDNAs were clonedfrom a 7-4 fetal liver stromal cell line cDNA library and Wnt-10b cDNAswere cloned using RT-PCR from flASK cell mRNAs. For the molecularcloning of Wnt cDNAs, poly A⁺ RNA was prepared by the Fast Track method(Invitrogen) and cDNA was made by denaturing PolyA⁺ RNA and dT₁₈ primersin the presence of 0.1 M methyl mercuric hydroxide, followed byquenching with 20 mM beta-mercaptoethanol and extension in 20 μl totalwith Superscript reverse transcriptase as recommended (Gibco).

A Wnt-5a PCR fragmentwas used to screen a cDNA library made from the 7-4cell line and ligated into the pSPORT-1 vector (Gibco). The Wnt-5acoding region was sequenced from one clone, Wnt5a.13.pSPORT-1, and thepredicted amino acids matched those previously reported (Gavin et al.,Genes and Dev., 4: 2319-2332 [1990]) except for (H207>Y). A Wnt-10b cDNAwas cloned by RT-PCR from flASK cells using primers wn10b.5ri (5'GGA ATTCCG GGC TTC GAC ATG CTG GAG GA 3') and wn10b.3kpn (5'GGG GTA CCC CAG GCTCAC CTT CAT TTA CAC A 3'), and cloned into pGEM7. The predicted codingsequence matched exactly to that reported elsewhere (Lee et al., Proc.Natl. Acad. Sci. USA, 92: 2268-2272 [1995]).

For overexpression of murine Wnts in mammalian cells and the preparationof conditioned media (CM) from the cells containing Wnt gene products,the Wnt-1, Wnt-5a, and Wnt-10b cDNAs thus obtained were cloned into asEcoRI/Hind III fragments into an expression vector pRK5tkneo (Holmes,Science, 253: 1278-1280 11991]). For production of CM, 293 cells weretransfected by the calcium phosphate method (Gorman, DNA Cloning: APractical Approach, IRL, Wash., D.C. [1985]), media was collected afterat least 48 hours, centrifuged at 3,000(SEQ ID NO: 20)×g, and sterilefiltered. In most experiments using CM, the CM was added to 5-10% finalconcentration. In preliminary experiments, most of the known cytokinesfailed to support the survival of flASK cells in suspension cultureswhen added as single factors in the presence of 10% fetal bovine serum(see, e.g., Bodine et al., Blood, 78: 914-920 [1991]). The addition ofKL at 100 ng/ml, however, provided a potent stimulus for cell survivaland proliferation of granulocytic progenitors (Anderson et al., Cell,63: 235-243 [1990]; Zsebo et al., Cell, 63: 195-201 [1990].Interestingly, control 293 CM provided an approximately 2-foldstimulatory activity when added to suspension cultures with KL. Theaddition of conditioned media (CM) from 293 cells transfected with aWnt-5a CDNA evoked a 2-3 fold greater expansion than control CM.

The presence of Wnt-5a protein in the CM from transfected cells wasconfirmed by immunoprecipitations and Western blotting. For theseexperiments, a chimeric Wnt-5a gene was made encoding the first 55 aminoacids (AA) of herpes simplex virus glycoprotein D followed by Wnt-5a AA38-379 in gDCT-1pRK5b (Pennica et al., Proc. Natl. Acad. Sci. USA, 92:1142-1146 [1995]). The control vector used in experiments involvinggDWnt5a was made from gDCT-1pRK5b by excising the CT-1 cDNA as aXhoI-Xbal fragment, filling in the overhanging nucleotides, and closingthe gDpRK5b vector with T4 DNA ligase (Collaborative Research). Theresulting construct was expected to encode the first 54 AA of gDfollowed by D stop. A fragment encoding six histidine residues wasappended in-frame to the carboxy terminus of Wnt-5a in the gDpRK5bvector by PCR.

For immunoprecipitation experiments for the analysis of Wnt expressionby transfected cells, 293 cells transfected with gDpRK5b or gDWnt5apRK5bwere labeled in 100 μCi [³⁵ S]methionine/[³⁵ S]cysteine (Amersham),lysed, and proteins were precipitated with the 5B6 mAb plus protein ASepharose® (Pharmacia). The precipitates were washed, electrophoresed,and fluorographed according to standard methods.

For comparison, the potent cytokines IL-3 and GM-CSF added as singlefactors to cultures with KL evoked a 1.5-2 fold greater expansion thancontrols. Further experiments revealed that titration of KL to 25 ng/mlprovided a lower expansion in cell number, but the cultures displayed areduced level of granulocytic proliferation/differentiation. Addition ofWnt-1, Wnt-5a, and Wnt-10b CM to the suspension cultures of flASK cellsplus 25 ng/ml KL stimulated cell proliferation 7, 8, and 11-fold(respectively) over that of control CM after 7 days in culture as shownin FIG. 1. A synergistic effect between KL and Wnts was evident sinceWnt CM alone triggered very little cell survival/proliferation. Thusfar, Wnt-1 expression has not been detected in the hematopoietic system,however, Wnt-1 CM was active in this assay. These results demonstratethe potential for distinct signaling pathways from several Wnt ligandsleading to a similar response of hematopoietic cells or the presence ofa common Wnt receptor in hematopoietic stem/progenitor cells.

B. Maintenance of the Blast Cell Phenotype and Foci Formation ofHematopoietic Progenitor Cells Enhanced by Wnts

In mice, Wnts are essential for the development of several primitivecell types and in Xenopus they are thought to be involved in cell fatedetermination. The role of Wnts was evaluated on the differentiationpotential of pluripotent hematopoietc stem/progenitor cells by examiningthe morphology of cultured cells in cytostained preparations. For thecytospin analysis, cells were grown in suspension culture as describedabove, spun onto a glass slide and then stained with Wright Geimsa toreveal hematopoietic cell types and morphology. For these experiments,cytospin analysis of flASK cells was performed with cytospinpreparations of (A) flask cells immediately after cell sorting, (B)flASK cells after suspension culture in control CM plus 25 ng/ml KL, and(C) flASK cells after suspension culture in Wnt-5a CM plus KL. Althoughaddition of KL to the suspension cultures was essential, even in reducedconcentrations, the effect of KL alone was to promote thedifferentiation and proliferation of granulocytic cells from HSCscompared to freshly isolated flASK cells. However, cells cultured inWnt-5a or Wnt-10b CM or partially purified recombinantWnt-5a (seeSection C below) generally gave rise to a greater diversity of celllineages with less commitmenttowards granulocytic lineages. Myeloidcells (macrophages and neutrophils), megakaryocytes, and early erythroidcells, were observed by cytospin analysis of suspension cultures aftertreatment with Wnt-5a CM. Notably, the ratio of primitive blasts todifferentiated mononuclear cells was elevated over 4-fold (29%±3.4compared to 7%±0.8 for the control) in cultures with recombinant Wnt-5a.The effect of Wnts plus KL was to promote extensive cell expansion, thatis the net increase in cell number from the initial HSC inoculum, whilealso maintaining a 4 fold greater proportion of cells with a primitiveblast cell morphology.

Wnts have been implicated in the regulation of cell adhesion systems,and it has been proposed that cell-cell interactions may be important incell fate determination,(Hinck et al., J. Cell. Biol,124: 729-741[1994]; Peiffer; Development Suppl., 163-176 [1993]). During the first4-5 days of suspension culture in Wnt CM, a dramatic increase in thenumber of loosely adherent cell aggregates or adherent hematopoietic`foci` were observed. To analyze the spatial organization and morphologyof these foci, flASK cells in HSC media with 25 ng/ml murine KL wereplated onto Lab-Tek glass chamber slides (NUNC, Naperville, Ill.) coatedwith 50 mg/ml of human plasma fibronectin (Gibco), cultured for 4-5 daysin control CM plus 25 ng/ml KL, and (E) Wnt-5a CM (gDWnt5a) plus 25ng/ml KL, and then cytostained in situ to preserve the intercellularorganization of the foci. Fibronectin was chosen as an adhesivesubstrate in this assay since it can mediate adhesion of CFU-Sprogenitors in vitro (Williams et al., Nature, 352: 438-441 [1991]).After 3-5 days in culture, clusters of 5 to more than 30 blasts with lowcytoplasm to nucleus ratios were typically found in contact with one ormore underlying adherent myeloid cells when the control culture inpreparation D was compared with Wnt-5a treated cells in preparation E).The formation of these blast cell foci was dramatically enhanced inresponse to Wnt CM and suggested a role for Wnts in cell expansion viathe regulation of cellular interactions.

In light of the enhanced proliferation and cell-cell adhesion of cellscultured in Wnt CM, the lineage phenotypes and adhesion systems of thecultured cells were analyzed by flow cytometry. For cell analysis andsorting experiments, phycoerythrin-conjugatedantibodies (Ly6A/E,TER-119, CD14), fluorescein-conjugatedantibodies (c-kit, CD13, CD31,CD44, CD45, CD49d, CD49e, GR1, VCAM-1, ICAM-1, L-selectin), CD11a, andCD29 antibodies were purchased from Pharmingen;phycoerythrin-conjugatedMac-1, fluorescein-conjugated antibodies (CD4,CD8a, and B220), and all secondary and Lin cocktail antibodies werepurchased from Caltag.

The flASK cells were cultured in gDWnt5a CM (Wnt CM) or gD CM (controlCM) for 7 days and scored for the expression of cell surface antigens.The expression of antigens found on mature hematopoietic cells (CD4,CD8a, CD13, CD14, B220, VCAM-1, and integrin beta-7) was low or negativeon freshly sorted flASK cells and remained at similar levels afterculture. Little or no change was observed in either condition forexpression of CD11a, CD29, CD31, CD44, CD45, CD49d, CD49e, GR1,L-selectin, ICAM-1. Cultures supplied with Wnt-5a CM did, however, havean increase in the number of cells that were Sca⁺ (154±22.6%), c-kit⁺(158±36%), Sca⁺ c-kit⁺ (131±9.4%), or Ter119⁺ (237±25.8%). These cellsurface antigen profiles compared well with the cytospin analysis andstrengthened the finding that Wnts plus KL promote the maintenance of agreater proportion of primitive blast cells than KL alone during the exvivo culture of HSCs.

C. Requirement of Secreted Wnt Protein in Wnt-Conditioned Media forProliferation of Hematopoietic Stem/Progenitor Cells

Several approaches were taken to distinguish a direct role of Wnts onHSCs from the alternative that Wnts stimulate the production of othergrowth factors in the transfected cells. Antibody depletion experimentswere carried out to confirm that the proliferation was mediated bysecreted Wnt proteins present in the media. An epitope-tag wasengineered onto Wnt-5a to enable depletion with readily availableantibody reagents. Chimeric proteins were constructed as described inSection A above that encoded the signal sequence and an N-terminaldomain of the herpes virus glycoprotein D (gD) followed by Wnt-5a(gDWnt5a, gDWnt5aHis₆. Transfection of the gDWnt5a construct into 293cells directed the expression of a 47-49kD polypeptide which wasspecifically immunoprecipitated from lysates by a mAb (5B6) recognizingan N-terminal epitope of gD. Immunodepletion experiments were conductedto remove the Wnt protein produced by the 293 transfected cells(gDWnt5apRK5b or control gDpRK5b) from the CM with the mAb5B6.Incubation of gDWnt5aHis₆ CM in mAb 5B6 bound to protein A or coupled tocontrolled-poreglass (5B6-CPG) reduced cell expansion to control values.Collectively, these data indicate that secreted Wnts mediated theobserved cell expansions in vitro. To test whether Wnts could directlystimulate cell proliferation, we evaluated the activity ofpartially-purifiedWnt-5a protein in the suspension culture assay.Preliminary experiments showed that cell extracts provided a richersource of Wnt-5a proteinthan CM. Forthe purification of gD.Wnt5a.His₆,stable lines of CHOdp12cells (DHFR cells, see, e.g., Bennett et al., J.Biol. Chem., 266: 23060 [1991]) that had been transfectedwith a DHFR⁺plasmid gD.Wnt5a.His₆ pSVi.del.d were selected and maintained inglutathione-S-transferase (GHT)-free media. Extracts were made fromgD.Wnt5a.His₆ CHO cells as follows. Cells were lysed in 50 mMTriethanolamine, 100 mM NaCI, 0.4% SDS, 1% PMSF, and 2% Triton® X-100.Purification of gD.Wnt5a.His₆ from the lysate was accomplished bybinding of the gD epitope to 5B6-CPG, extensive washing in PBS, and acidelution. The eluate was neutralized, dialyzed against PBS, and refoldedin 8 M urea. The refolded Wnt-5a protein was diluted in HSC media sothat concentration of urea was less than 60 mM Wnt-5a in the stem cellsuspension culture assay.

Gel analysis revealed that the protein migrated as a 47-49kD monomerunder reducing conditions. When added to the suspension culture assay,recombinant Wnt-5a protein was found to stimulate cell expansion by5-fold at approximately 40-80 ng/ml or 1-2 nM.

D. Stimulation of Total CFC Expansion of Hematopoietic Progenitor Cellsby Wnt Protein

An important measure of hematopoietic progenitor cells is the ability toform colonies in semi-solid media in response to lineage-specificcytokines and multilineage colony-stimulating factors. Since theaddition of Wnts plus KL to flASK cell cultures increased the proportionof cells with a primitive morphology and cell surface phenotype,experiments were performed to analyze whether the number of highlyproliferative colony-forming cells had increased as well. The frequencyof colony forming cells (CFCs) in flASK cell cultures was examined bymeasuring colony formation of cells replated into myeloidmethylceliulose containing a combination of cytokines (KL, IL-3, IL-6,and Epo) and Wnt CM or control CM. For the colony assay experiments,methylcellulose cultures were initiated in 35 mM plates with 1000 cellsin 1.0 ml complete myeloid methylcellulose (Stem Cell Technologies,Inc.) or in B-cell conditions consisting of base methylcellulosecontaining 50 ng/ml murine KL and 50 ng/ml murine IL-7 (R&D Systems).Conditioned media was added at the time of plating. Plates were read atday 12 after plating. After suspension culture, the total CFCs derivedfrom 1000 cells in the initial culture inoculum was 3-fold greater inWnt-5a CM than control CM. Moreover, when cells were harvested from day12 myeloid methylcellulose cultures and replated, the cumulativeexpansion of cells brought forth by Wnt-5a CM was over 235 fold greaterthan for control CM, largely due to the inability of cells grown incontrol CM to efficiently replate. These results provide compellingfunctional evidence that secreted Wnts enhance thesurvival/proliferation of multipotent hematopoietic progenitors insuspension culture and can directly stimulate the expansion ofprimitive, highly proliferative colony forming cells.

Colony formation of freshly isolated fetal liver AA4⁺ Sca⁺ cells inWnt-5a CM was also tested. Wnt-5a CM stimulated colony formationapproximately 3-fold in myeloid methylcellulose. In B-cell conditions,Wnt-5a CM stimulated colony formation 4-fold. Colony formation wasenhanced 2-fold for bone marrow Lin^(lo) Sca+ cells in myeloid andB-cell methylcellulose. Overall, these data show that Wnt CM enhancescolony formation by highly enriched fetal liver and bone marrow HSCs inconditions that detect myeloid or B-cell progenitors.

E. Expansion of Hematopoietic Stem/Progenitor Cells After Transductionwith a Retrovirus Bearing a Wnt Protein

To further examine the direct effects of Wnt expression on HSCs, arepresentative Wnt protein product, Wnt-5a, was introduced viaretroviral transduction. A Rous sarcoma virus-based bicistronic LNL6vector was constructed so that Wnt-5a was placed 3' to the gag gene andLacZ was downstream of the encephalomyocarditis virus internal ribosomeentry site. Specifically, for the viral construction and transductionexperiments, Wnt5a.13.pSPORT-1 was digested with EcoRI/BamHl, the insertwas blunted with T4 DNA Polymerase (U.S. Biochemical), and cloned intoblunted Bg/II/BamHl sites of the pLNL6 vector (Ghattas et al., Mol.Cell. Biol., 11: 5848-5859 [1991]). Wnt5a/LNL6 or the parental LNL6vectorwere transfected by a calcium phosphate method into BOSC 23 cells(Pear et al., Proc. Nati. Acad. Sci. USA, 90: 8392-8396 [1993]). Viralsupernatantsfrom the transfected BOSC cells were collected after 48-72hours and stored at -20° C and used to transduce flASK cells.Transductions of the flASK cells were carried out at 100,000 cells perml for 48 hours in viral supernatants supplemented with the murinecytokines IL-3 (25 ng/ml), IL-6 (50 ng/ml), and KL (10 ng/ml).Transduction efficiency of cells giving rise to methylcellulose colonieswas assayed by PCR analysis essentially as described in Gerard et al.,Human Gene Therapy, 7: 343-354 [1996]. Expression of the biscistronicmRNA in transduced flASK cells was confirmed by measuring LacZ activityusing the FACS-Gal method (Fiering et al., Cytometry, 12: 291-301[1991]). The added cytokines IL-3, IL-6, and KL increased thetransduction efficiency, which was estimated to be approximately 20% byFACS-Gal analysis 48 hours post-transduction. A potential early-actingeffect of Wnt-5a on cell survival/proliferation was measured by countingcell numbers 48 hours after transduction. Wnt5a/LNL6-transduced cellsexpanded by almost 2-fold (FIG. 2A). Notably, this cellsurvival/proliferation was impressive in that an additional benefit wasobserved over the potent effects of the early-acting cytokines IL-3,IL-6, and KL. Culture of the Wnt5a/LNL6-transduced cells for 7 daysrevealed extensive proliferation compared to that of control cells (FIG.2B). Cells from 2 day transductions were also replated into myeloidmethylcellulose. Transduction with Wnt5a/LNL6 stimulated a 3-foldgreater expansion of CFCs than the control vector (FIG. 2C). Theefficiency of CFC transduction was estimated to be 14-47% by PCRanalysis of colonies plucked from the methylcellulose cultures.

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    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 12 base                                                           (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #       12                                                                    - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                # 20               GCTC                                                       - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                # 20               CCGT                                                       - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #                 22CCT CG                                                    - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 22 base                                                           (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #                 22TTG CG                                                    - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #               25 TGCA AATGC                                                 - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #               25 ACAC ATTGA                                                 - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #            29    ACAT GCTGGAGGA                                             - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #          31      ACCT TCATTTACAC A                                          __________________________________________________________________________

What is claimed is:
 1. A method for enhancing or increasingmyelopoiesis, erythropoiesis or lymphopoiesis comprising exposinghematopoietic stem/progenitor cells to an amount of isolated Wntpolypeptide effective for stimulating proliferation or differentiationof the cells.
 2. The method of claim 1, wherein the cells comprise CD34⁺cells.
 3. The method of claim 1, wherein the cells comprise AA4⁺ cells.4. The method of claim 1, wherein the cells comprise flASK cells.
 5. Themethod of claim 1, wherein the hematopoietic stem/progenitor cells arepresent in cell culture.
 6. The method of claim 1, wherein the cells arepresent in a mammal.
 7. The method of claim 6, wherein the mammal is ahuman.
 8. The method of claim 1, which enhances or increaseslymphopoiesis.
 9. The method of claim 1, which enhances or increaseserythropoiesis.
 10. The method of claim 1, which enhances or increasesmyelopoiesis.
 11. The method of claim 1, further comprising exposing thecells to another cytokine.
 12. The method of claim 11, wherein thecytokine is lineage-specific.
 13. The method of claim 1, wherein thecytokine is selected from the group consisting of: thrombopoietin (TPO),erythropoietin (EPO), macrophage-colony stimulating factor (M-CSF),granulocyte-macrophage-colony stimulating factor (GM-CSF),granulocyte-colony stimulating factor (G-CSF), interleukin-1 (IL- 1),IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, leukemia inhibitory factor and kit ligand.